Multi-arm polymeric alkanoate conjugates

ABSTRACT

Among other aspects, provided herein are multi-armed polymer conjugates comprising an alkanoate-linker, compositions comprising such conjugates, and related methods of making and administering the same. Methods of treatment employing such conjugates and related uses are also provided. The conjugates are prepared with high drug loading efficiencies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/058,320, filed May 6, 2011, which is a 35 U.S.C. §371 application ofInternational Application No. PCT/US2009/004618, filed Aug. 11, 2009,designating the United States, which claims the benefit of priority to:Provisional Patent Application No. 61/087,826, filed Aug. 11, 2008;Provisional Patent Application No. 61/106,928, filed Oct. 20, 2008; andProvisional Application No. 61/113,328, filed 11 Nov. 2008; the contentsof which are each hereby expressly incorporated herein by reference intheir entireties.

FIELD

This disclosure relates generally to multi-arm, water-soluble polymersand their corresponding drug conjugates. In particular, the disclosureis directed to, among other features, multi-armed polymer conjugateshaving drug covalently attached to the multi-arm polymer via analkanoate-linkage. Also disclosed are pharmaceutical compositionscomprising such conjugates, and methods for preparing, formulating,administering, and using such conjugates and related compositions.

BACKGROUND

Over the years, numerous methods have been proposed for improving thedelivery of biologically active agents, particularly small moleculedrugs. Challenges associated with the formulation and delivery ofpharmaceutical agents can include poor aqueous solubility of thepharmaceutical agent, toxicity, low bioavailability, instability, andrapid in-vivo degradation, to name just a few. Although many approacheshave been devised for improving the delivery of pharmaceutical agents,no single approach is without its drawbacks. For instance, commonlyemployed drug delivery approaches aimed at solving or at leastameliorating one or more of these challenges include drug encapsulation(such as in a liposome, polymer matrix, or unimolecular micelle),covalent attachment to a water-soluble polymer (i.e., conjugation) suchas polyethylene glycol (i.e., PEG or PEGylation), use of gene targetingagents, and the like.

PEGylation has been employed to a limited degree to improve thebioavailability and ease of formulation of small molecule therapeuticshaving poor aqueous solubilities. For instance, water-soluble polymerssuch as PEG have been covalently attached to artilinic acid to improveits aqueous solubility. See U.S. Pat. No. 6,461,603. Similarly, PEG hasbeen covalently attached to triazine-based compounds such as trimelamolto improve their solubility in water and enhance their chemicalstability. See International Patent Application Publication No. WO02/043772. Covalent attachment of PEG to bisindolyl maleimides has beenemployed to improve poor bioavailability of such compounds due to lowaqueous solubility. See International Patent Application Publication No.WO 03/037384). Polymer conjugates of non-steroidal anti-inflammatorydrugs (NSAIDs) and of opioid antagonists have also been prepared. SeeU.S. Patent Application Publication Nos. 2007/0025956 and 2006/0105046,respectively. Prodrugs of camptothecin having one or two molecules ofcamptothecin covalently attached to a linear polyethylene glycol havealso been prepared. See U.S. Pat. No. 5,880,131.

Certain drugs, such as the alkaloids, are notoriously difficult tosolubilize (i.e., formulate). Such alkaloids include the taxanes, suchas docetaxel, and the camptothecins, such as irinotecan. Camptothecin(often abbreviated as “CPT”) is a phytotoxic alkaloid first isolatedfrom the wood and bark of Camptotheca acuminata (Nyssaceae), and hasbeen shown to exhibit antitumor activity. The compound has a pentacyclicring system with an asymmetric center in lactone ring E with a 20 Sconfiguration. The pentacyclic ring system includes apyrrolo[3,4-b]quinoline (rings A, B and C), a conjugated pyridone (ringD), and a six-membered lactone (ring E) with a 20-hydroxyl group. Due toits insolubility in water, camptothecin was initially evaluatedclinically in the form of a water-soluble carboxylate salt having thelactone ring open to form the sodium salt. The sodium salt, althoughexhibiting much improved water solubility in comparison to camptothecinitself, produced severe toxicity and demonstrated very little in vivoanticancer activity, thus demonstrating the undesirability of thisapproach. A Phase I clinical trial for a linear PEG-paclitaxel compoundfor treatment of patients with advanced solid tumors and lymphomas in2001; the trial has since been terminated.

In an effort to address the poor aqueous solubility associated withcamptothecin and many of its derivatives, a number of synthetic effortshave been directed to derivatizing the A-ring and/or B-ring oresterifying the 20-hydroxyl to improve water-solubility whilemaintaining cytotoxic activity. For example, topotecan(9-dimethylaminomethyl-10-hydroxy CPT) and irinotecan(7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxy CPT), otherwiseknown as CPT-11, are two water-soluble CPT derivatives that have shownclinically useful activity. Conjugation of certain camptothecinderivatives, such as 10-hydroxycamptothecin and 11-hydroxycamptothecin,to a linear poly(ethylene glycol) molecule via an ester linkage has beendescribed as a means to form water soluble prodrugs. See U.S. Pat. No.6,011,042. The approach used relies on reaction of an aromatic,hydroxyl-containing compound with an activated polymer.

The clinical effectiveness of many small molecule therapeutics such asthe foregoing, and oncolytics in particular, is limited by severalfactors such as dose-related toxicity. For instance, irinotecan andother camptothecin derivatives undergo an undesirable hydrolysis of theE-ring lactone under alkaline conditions. Additionally, administrationof irinotecan causes a number of troubling side effects, includingleukopenia, neutropenia, and diarrhea. Due to its severe diarrhealside-effect, the dose of irinotecan that can be administered in itsconventional, unmodified form is extremely limited, thus hampering theefficacy of this drug and others of this type.

These associated side effects, when severe, can be sufficient to arrestfurther use as well as development of such drugs as promisingtherapeutics. Additional challenges facing small molecules include highclearance rates, and, with respect to anticancer agents, minimal tumorpermeation and residence time. Approaches involving the use of polymerattachment must balance the size of the polymer against the molecularweight of the active agent in order to allow therapeutically effectivedoses to be delivered, and at a clinically useful rate. Finally, thesynthesis of a modified or drug delivery-enhanced active agent mustresult in reasonable yields, and in a reproducibly prepared product, tomake any such approach economically attractive. Thus, there exists aneed for new methods for effectively delivering drugs, and in particularsmall molecule drugs, and even more particularly oncolytics, which canreduce their adverse and often toxic side-effects, whilst simultaneouslyimproving their preparation, efficacy and ease of formulation. Even moreimportantly, there exists a need to provide oncolytic products that areeffective against drug resistant tumors. Specifically, there is a needfor improved methods for delivering drugs such as the foregoing thatpossess an optimal balance of bioavailability due to reduced clearancetimes, bioactivity, and efficacy, coupled with reduced side-effects.

SUMMARY

In a first aspect, the present disclosure provides a multi-arm polymerconjugate having the following structure,

where: R is an organic core radical comprising from about 3 to about 150carbon atoms; Q is a linker; POLY₁ is a water-soluble and non-peptidicpolymer segment; X is a spacer that is optionally present; R₁, in eachoccurrence, is independently selected from the group consisting of H,lower alkyl, and an electron withdrawing group; n is an integer in therange from 1 to 7 (e.g., is selected from 1, 2, 3, 4, 5, 6, and 7); D isa residue of a small molecule having a molecular weight of less thanabout 800 daltons; and q is 3 or greater.

Each of the following embodiments described herein may be consideredsingly, or taken in combination with any one or more additionalembodiments, so long as the particular combination is not mutuallyinconsistent with the particular embodiments included in suchcombination.

In particular embodiments in reference to Compound I, D is an anticanceragent.

In yet other embodiments, the variable “R” is an organic core radicalpossessing from about 3 to about 25 carbon atoms. In yet furtherembodiments, R can be linear or cyclic. In additional embodiments, R isa saturated, aliphatic core. Exemplary multi-armed polymer conjugatesinclude those where R, when taken together with Q over each of “q” arms,is a residue of a polyol, a polythiol, or a polyamine. In one or moreparticular embodiments, R, when taken together with Q over each of “q”arms, is a residue of glycerol, trimethylolpropane, pentaerythritol,sorbitol, or an oligomer of glycerol.

In yet other embodiments, linker, “Q,” is hydrolytically stable.Particular embodiments related to Q include the following: in one ormore embodiments, Q contains from about 1 to about 10 atoms; in otherembodiments, Q is selected from the group consisting of —O—, —S—, —NH—,—C(O)—NH—, and —NH—C(O)—.

In yet other embodiments, POLY₁ is a polymer selected from the groupconsisting of poly(alkylene glycol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharide), poly(α-hydroxy acid),poly(acrylic acid), polyvinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), or copolymers or terpolymers thereof.

In preferred embodiments, POLY₁ is a polyethylene glycol.

Additional embodiments directed to POLY₁ include the following: in oneor more embodiments, POLY₁ is linear; in yet other embodiments, theweight average molecular weight of POLY₁ ranges from about 200 to about30,000 daltons.

In further embodiments, the weight average molecular weight of theconjugate is about 20,000 daltons or greater, e.g., in a molecularweight range of about 20,000 to about 80,000 daltons.

Particular embodiments directed to variable “X” (which is optionallypresent) include the following. In one or more embodiments, X has anatom length of from about 1 atom to about 50 atoms. In yet otherembodiments, X has an atom length of from about 1 atom to about 25 atoms(e.g., is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range therein). Infurther embodiments, X is oxygen (“—O—”). In a particular embodimentwhen POLY₁ is a linear polyethylene glycol corresponding to thestructure, —(CH₂CH₂O)_(n)CH₂CH₂—, X is oxygen (“—O—”).

Additional embodiments related to the variable “R₁” include thefollowing. In one or more embodiments, R₁ is H. In yet otherembodiments, R, is H for all “n” occurrences. In yet other embodiments,each R₁ in each occurrence [i.e., for each of “n” of (˜C(H)R₁˜)] isindependently selected from methyl, ethyl, propyl, n-butyl, isopropyland isobutyl. In yet other embodiments, each R, is independentlyselected from a halide (e.g., fluoride, chloride, bromide, and iodide),a nitrile, —NO₂, and —CF₃. In preferred embodiments, R₁ is hydrogen. Inyet other embodiments, R₁ is other than hydrogen only when positioned onthe alpha carbon adjacent to the carbonyl. In further embodiments, R₁ ismethyl when positioned on the alpha carbon.

Turning now to the variable “q,” particular embodiments directed to “q”include the following. In one or more embodiments, the value of q isselected from the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10. Inyet other embodiments, q is 3 or 4. In yet other embodiments, each of“q” polymer arms [-Q-POLY₁-X—(CH₂)₂—(CHR₁)_(n)C(O)OD] is the same.

Embodiments directed to the small molecule portion of the conjugateinclude the following. In one or more embodiments, the small moleculepossesses a molecular weight ranging from about 250-700 daltons. In yetother embodiments, D is a residue of a taxane or a camptothecin. In yetfurther embodiments, D is a residue of paclitaxel or docetaxel. In oneor more embodiments, D possesses the structure:

In yet a further embodiment, the multi-armed polymer conjugate possessesthe structure:

where n ranges from about 40 to about 500. In an embodiment related tothe foregoing structure, the overall weight average molecular weight ofthe conjugate ranges from about 10,000 to about 80,000.

In a second aspect, provided is a pharmaceutical composition comprisinga multi-armed polymer conjugate as described herein combined with apharmaceutically acceptable carrier.

In a related embodiment in which the multi-armed polymer conjugatecorresponds to Compound Ia shown above, the composition, when evaluatedin a single dose study in rats, exhibits a 2-fold or greater reductionof toxicity when compared to docetaxel. In yet other embodiments, theconjugate is characterized by a drug loading of greater than or equal to92%. In yet other embodiments, the multi-armed polymer conjugate, whichcorresponds to Compound Ia shown above, is further characterized by apercentage tumor growth delay (TGD) when measured at maximum tolerateddose (MTD) in any one of a H460, LoVo, or LS 174T mouse xenograft model,that is 1.5-fold or more greater than that observed for docetaxel.

In a third aspect, provided herein is a method of delivering amulti-armed polymer conjugate to a mammalian subject in need thereof.The method comprises administering to the mammalian subject atherapeutically effective amount of a multi-armed polymer conjugate asprovided herein.

In a fourth aspect, provided herein is a method of treating cancer in amammalian subject. The method comprises administering a therapeuticallyeffective amount of a conjugate as provided herein (where D is ananticancer agent) to a subject diagnosed as having one or more canceroussolid tumors over a duration of time effective to produce an inhibitionof growth of said solid tumor(s) in said subject.

In an embodiment directed to the foreoing fourth aspect, the canceroussolid tumor type is selected from the group consisting of colorectal,breast, prostate, and non-small cell lung.

In a fifth aspect, provided herein is a method of treating a mammaliansubject for a condition responsive to treatment with docetaxel, wherethe method comprises administering to the subject a therapeuticallyeffective amount of a multi-armed polymer conjugate as provided herein.

Also provided herein are uses related to the foregoing methods.

In a sixth aspect, in a multi-armed polymer conjugate having thestructure,

R(-Q-POLY₁-Y-D)_(q),

where R is an organic core radical possessing from about 3 to about 150carbon atoms, Q is a linker, POLY₁ is a water-soluble and non-peptidicpolymer, Y is a spacer comprising a hydrolyzable linkage, such that uponhydrolysis of said hydrolyzable linkage, D is released, D is a smallmolecule, and q is greater than or equal to 3, provided is animprovement comprising Y having the structure,

where: X is a spacer as described in further detail herein; R₁ isselected from the group consisting of H, lower alkyl, and an electronwithdrawing group, and n is an integer from 1 to 5.

In a seventh aspect, provided is a multi-arm polymer structure havingthe following structure,

where: R is an organic core radical comprising from about 3 to about 150carbon atoms; Q is a linker; POLY₁ is a water-soluble and non-peptidicpolymer segment; X is a spacer; each R₁ is independently selected fromthe group consisting of H, lower alkyl, and an electron withdrawinggroup; n is an integer from 1 to 7; q is 3 or greater; W is selectedfrom the group consisting of D, H, and an activated ester, where D is aresidue of a small molecule having a molecular weight of less than about800 daltons. With respect to compositions comprising this multi-armpolymer structure, on average and taking into account all species withinthe composition, W is preferably equal to D at a value of 0.92(q) orgreater.

In an eighth aspect, provided is a method of preparing a multi-armpolymer drug conjugate. The method comprises reacting within the limitsof the described chemical processes a multi-arm water-soluble polymerstructure having “q” polymer arms, each having a reactive carboxylicacid group or activated ester equivalent at its terminus, with “q”equivalents or greater of a sterically hindered secondary or tertiaryalcohol of a small molecule drug under conditions effective to result inconjugation of the small molecule drug to the multi-arm water solublepolymer structure via an ester linkage to form a multi-arm water solublepolymer drug conjugate wherein 92% or greater of said polymer arms havesmall molecule drug covalently attached thereto. The conjugate possessesa linker adjacent to the ester linkage in each of the polymer arms,where the linker is preferably absent a functional group capable of aneighboring group interaction with the ester carbonyl to form a five- orsix-membered cyclic product capable of displacing the small moleculedrug.

In a particular embodiment related to the eighth aspect, the linker isan alkanoate linker having the structure —(CH₂)₂(CR₁H)_(n)C(═O)—O—,where R₁ in each occurrence is independently selected from H, loweralkyl, and an electron withdrawing group, and n is an integer from 1 to7. In yet another embodiment, prior to the reacting step, the linker iscovalently attached to the drug or to each of the polymer arms.

In yet a ninth aspect, provided herein is a multi-arm polymer conjugateor reagent having the following structure:

where each of the variables shown are as described above. In oneparticular embodiment of the foregoing, the structure possesses onemethylene group adjacent to X rather than two, n is equal to one, and R₁is either lower alkyl or an electron withdrawing group.4-arm-PEG-methylpropionic acid is an exemplary structure that is usefulin providing such multi-arm polymer conjugates or reagents.

Additional embodiments of the present method, compositions, and the likewill be apparent from the following description, drawings, examples, andclaims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

These and other objects and features of the invention will become morefully apparent when read in conjunction with the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C demonstrate an illustrative synthesis of a dipeptide-linkedpentaerythritolyl core-containing multi-armed polymer.

FIGS. 2A and 2B are graphs illustrating the median tumor volume (MTV)over time for mice implanted with LS 174T (colorectal) tumors andtreated with either docetaxel (Taxotere®) or 4-ARM-PEG_(20K)-BA-DOC asdescribed in detail in Example 13. FIG. 2A provides dose response curvesfor mice administered different dosages of either docetaxel or4-ARM-PEG_(20K)-BA-DOC; FIG. 2B is a graphical representation of LS 174Tmedian tumor volume over time for mice administered the maximumtolerated dose (MTD) of either docetaxel or 4-ARM-PEG_(20K)-BA-DOC.

FIGS. 3A and 3B illustrate significantly increased tumor growth delay(TGD) for mice implanted with H460 tumors and treated with eitherdocetaxel (Taxotere®) or 4-ARM-PEG_(20K)-BA-DOC as described in detailin Example 13. FIG. 3A provides dose response curves for miceadministered different dosages of either docetaxel or4-ARM-PEG_(20K)-BA-DOC; FIG. 3B provides a maximum tolerated dose (MTD)versus control comparison.

FIGS. 4A and 4B are graphs illustrating the median tumor volume overtime for mice implanted with LoVo tumors and treated with eitherdocetaxel (Taxotere®) or 4-ARM-PEG_(20K)-BA-DOC as described in detailin Example 13. FIG. 4A provides dose response curves for miceadministered different dosages of either docetaxel or4-ARM-PEG_(20K)-BA-DOC; FIG. 4B provides a MTD comparison for miceadministered the maximum tolerated dose (MTD) of either docetaxel or4-ARM-PEG_(20K)-BA-DOC versus control.

DETAILED DESCRIPTION

Various aspects of the invention now will be described more fullyhereinafter. Such aspects may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

DEFINITIONS

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

A “functional group” is a group that may be used, under normalconditions of organic synthesis, to form a covalent linkage between theentity to which it is attached and another entity, which typically bearsa further functional group. The functional group generally includesmultiple bond(s) and/or heteroatom(s). Preferred functional groups foruse in the polymers of the invention are described below.

The term “reactive” refers to a functional group that reacts readily orat a practical rate under conventional conditions of organic synthesis.This is in contrast to those groups that either do not react or requirestrong catalysts or impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

“Not readily reactive”, with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters include, forexample, imidazolyl esters, and benzotriazole esters, and imide esters,such as N-hydroxysuccinimidyl (NHS) esters. An activated derivative maybe formed in situ by reaction of a carboxylic acid with one of variousreagents, e.g. benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in combination with1-hydroxy benzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT);O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); or bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOP-Cl).

A “chemical equivalent” of a functional group is one that possessesessentially the same type of reactivity as the functional group. Forinstance, one functional group that undergoes an SN2 reaction isconsidered to be a functional equivalent of another such functionalgroup.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups thatmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andin P. J. Kocienski, Protecting Groups, Third Ed., Thieme Chemistry,2003, and references cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)-” or “—(CH₂CH₂O)_(n-1) CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation, or, e.g., the identity of adjacent functionalgroups. The variable (n) typically ranges from 3 to about 3000, and theterminal groups and architecture of the overall PEG may vary. When PEGor a conjugate comprising a PEG segment further comprises a spacer or alinker as in Compound I above (to be described in greater detail below),the atoms comprising the spacer (X) or linker (Q), when covalentlyattached to a PEG segment, do not result in formation of (i) anoxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N). PEGs for use in the invention includePEGs having a variety of molecular weights, structures or geometries tobe described in greater detail below.

“Water-soluble,” in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

An “end-capping” or “end-capped” group is an inert group present on aterminus of a polymer such as PEG. An end-capping group is one that doesnot readily undergo chemical transformation under typical syntheticreaction conditions. An end capping group is generally an alkoxy group,—OR, where R is an organic radical comprised of 1-20 carbons and ispreferably lower alkyl (e.g., methyl, ethyl) or benzyl. For instance, anend capped PEG will typically comprise the structure“RO—(CH₂CH₂O)_(n)—”, where R is as defined above. Alternatively, theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled, can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

“Non-naturally occurring” with respect to a polymer means a polymer thatin its entirety is not found in nature. A non-naturally occurringpolymer may however contain one or more subunits or segments of subunitsthat are naturally occurring, so long as the overall polymer structureis not found in nature.

“Molecular mass” in the context of a water-soluble polymer such as PEG,refers to the nominal average molecular mass of a polymer, typicallydetermined by size exclusion chromatography, light scatteringtechniques, or intrinsic viscosity determination in water or organicsolvents. Molecular weight in the context of a water-soluble polymer,such as PEG, can be expressed as either a number-average molecularweight or a weight-average molecular weight. Unless otherwise indicated,all references to molecular weight herein refer to the number-averagemolecular weight. Both molecular weight determinations, number-averageand weight-average, can be measured using gel permeation chromatographictechniques. Other methods for measuring molecular weight values can alsobe used, such as the use of end-group analysis or the measurement ofcolligative properties (e.g., freezing-point depression, boiling-pointelevation, or osmotic pressure) to determine number-average molecularweight or the use of light scattering techniques, ultracentrifugation orviscometry to determine weight-average molecular weight. The polymers ofthe invention are typically polydisperse (i.e., number-average molecularweight and weight-average molecular weight of the polymers are notequal), possessing low polydispersity values such as less than about1.2, less than about 1.15, less than about 1.10, less than about 1.05,and less than about 1.03. As used herein, references will at times bemade to a single water-soluble polymer having either a weight-averagemolecular weight or number-average molecular weight; such referenceswill be understood to mean that the single-water soluble polymer wasobtained from a composition of water-soluble polymers having the statedmolecular weight.

The term “linker” is used herein to refer to an atom or a collection ofatoms used to link interconnecting moieties, such as an organic radicalcore and a polymer segment, POLY₁. A linker moiety may be hydrolyticallystable or may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

The term “spacer” is used herein to refer to an atom or a collection ofatoms used to link interconnecting moieties, such as POLY₁ and adimethylene group forming part of an alkanoate linkage to small moleculedrug, D. A spacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage.

A “hydrolysable” bond is a relatively weak bond that reacts with water(i.e., is hydrolyzed) under physiological conditions. The tendency of abond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Illustrative hydrolytically unstablelinkages include carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes. Such a linkage requires the actionof one or more enzymes to effect degradation.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Multi-armed” in reference to the geometry or overall structure of apolymer refers to polymer having 3 or more polymer-containing “arms”connected to a “core” molecule or structure. Thus, a multi-armed polymermay possess 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymerarms, 7 polymer arms, 8 polymer arms or more, depending upon itsconfiguration and core structure. One particular type of highly branchedpolymer is a dendritic polymer or dendrimer, that, for the purposes ofthe invention, is considered to possess a structure distinct from thatof a multi-armed polymer. That is to say, a multi-armed polymer asreferred to herein explicitly excludes dendrimers. Additionally, amulti-armed polymer as provided herein possesses a non-crosslinked core.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers are typically formed using a nano-scale, multistepfabrication process. Each step results in a new “generation” that hastwo or more times the complexity of the previous generation. Dendrimersexhibit certain dendritic state properties such as core encapsulation,making them unique from other types of polymers.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms. A multi-arm polymer may have onebranch point or multiple branch points, so long as the branches are notregular repeats resulting in a dendrimer.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, isopropyl, n-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl,3-methyl-3-pentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or Spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl (vinyl), 2-propen-1-yl (allyl), isopropenyl,3-buten-1-yl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, 1-propynyl, 3-butyn-1-yl, 1-octyn-1-yl, and soforth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion, atom, or collection of atoms that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center, and capable of reacting with an electrophile.

“Active agent” as used herein includes any agent, drug, compound, andthe like which provides some pharmacologic, often beneficial, effectthat can be demonstrated in vivo or in vitro. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of an active agent present in a pharmaceuticalpreparation that is needed to provide a desired level of active agentand/or conjugate in the bloodstream or in a target tissue or site in thebody. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multi-functional” in the context of a polymer of the invention means apolymer having 3 or more functional groups, where the functional groupsmay be the same or different, and are typically present on the polymertermini. Multi-functional polymers of the invention will typicallycontain from about 3-100 functional groups, or from 3-50 functionalgroups, or from 3-25 functional groups, or from 3-15 functional groups,or from 3 to 10 functional groups, i.e., contains 3, 4, 5, 6, 7, 8, 9 or10 functional groups. Typically, in reference to a polymer precursorused to prepare a polymer conjugate of the invention, the polymerpossesses 3 or more polymer arms having at the terminus of each arm afunctional group suitable for coupling to an active agent moiety via ahydrolyzable ester linkage. Typically, such functional groups are thesame.

“Difunctional” or “bifunctional” as used interchangeable herein means anentity such as a polymer having two functional groups contained therein,typically at the polymer termini. When the functional groups are thesame, the entity is said to be homodifunctional or homobifunctional.When the functional groups are different, the polymer is said to beheterodifunctional or heterobifunctional.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

The terms “subject,” “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals and pets. Such subjects are typically suffering from orprone to a condition that can be prevented or treated by administrationof a polymer of the invention, typically but not necessarily in the formof a polymer-active agent conjugate as described herein.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Treatment” or “treating” of a particular condition includes: (1)preventing such a condition, i.e. causing the condition not to develop,or to occur with less intensity or to a lesser degree in a subject thatmay be exposed to or predisposed to the condition but does not yetexperience or display the condition, (2) inhibiting the condition, i.e.,arresting the development or reversing the condition.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

A “small molecule” may be defined broadly as an organic, inorganic, ororganometallic compound typically having a molecular weight of less thanabout 1000, preferably less than about 800 daltons. Small molecules ofthe invention encompass oligopeptides and other biomolecules having amolecular weight of less than about 1000.

A “residue,” for example, of a small molecule (in reference to aconjugate as provided herein, refers to the portion or residue of theumodified small molecule up to the covalent linkage resulting fromcovalent attachment of the small molecule (or an activated or chemicallymodified form thereof) to a multi-armed polymer as provided herein. Forexample, upon hydrolysis of the hydrolyzable ester linkage between thesmall molecule moiety and the multi-armed polymer, the small moleculeper se is released. For example, for a small molecule drug comprising ahydroxyl group and represented by the informal formula, D-OH, inreference to Compound I, the residue of the small molecule willcorrespond to “D,” which is the portion of the small molecule remainingupon removal or replacement of the hydroxyl group, —OH. In reference tothe organic core molecule, “R,” the following example is provided. Inthe instance in which R is a residue of a molecule possessing threehydroxyls (a triol), represented as R—(OH)₃, when considered inreference to Compound I, then R—(O˜) for each polymer arm, q, isconsidered as the residue of the polyol, where R is considered incombination with linker, Q, to be a residue of a triol. In the precedingexample, oxygen (O) corresponds to linker, Q.

A “polyol” is an alcohol containing more than two hydroxyl groups, wherethe prefix “poly” in this instance refers to a plurality of a certainfeature rather than to a polymeric stucture. Similarly, a polythiol is athiol containing more than two thiol (—SH) groups, and a polyamine is anamine containing more than two amino groups.

Multi-Arm Polymer Conjugates—Neighboring Group Interactions and theirAvoidance

The multi-armed polymer conjugates provided herein are also referred toas “prodrugs.” The term “prodrug” refers, in the instant application, toa modified form of a drug, where such modification typically includes acovalent link between the drug and a chemical moiety (e.g., a polymer).A prodrug is converted in vivo to its active drug form, eitherenzymatically or nonenzymatically.

Multi-armed polymer conjugates of small molecules have been previouslydescribed. See, e.g., U.S. Patent Application Publication Nos.2005/0112088 and 2007/0197575. Certain exemplified embodiments of themulti-armed polymer conjugates described therein contain an amino acidlinker such as glycine connecting the active agent, e.g., a camptothecinmolecule, to the multi-armed polymer scaffold. An amino acid linkagesuch as glycine is attractive due to its bifunctional nature and facilehydrolysis in vitro and in vivo. The ester linkage resulting from theamino acid linker is rapidly hydrolyzed due to the activation of theelectron withdrawing nature of the functional group adjacent to theglycine moiety. However, although such a linker can provide advantageoushydrolysis rates, the inventors have discovered that a neighboringfunctional group interaction may cause problems under certaincircumstances which may make such linkers sub-optimal in the finalmulti-arm polymer conjugates.

As a result of understanding how neighboring group processes occur (see,e.g., Capon and McManus, Neighboring Group Participation, Plenum Press,New York, 1975) and recognizing their potential occurrence, theinventors have fashioned (among other things) new conjugates, reagents,and synthetic schemes with this understanding in mind. Among otherthings, the present multi-armed polymer conjugates circumvent and/orreduce a neighboring group interaction that can result in azlactoneformation—leading to less than optimal drug loading in the finalmulti-arm polymer drug conjugate. An azlactone is a compound that is ananhydride of an α-acylamino acid; the basic ring structure is the5-oxazolone type, e.g.,

An azlactone can form, for example, in the presence of a condensingagent, when attempting to form an active ester of a carboxylic acid.

Azlactones can form in many similar processes. See, e.g., Benoiton etal. (1981) “Oxazolones from 2-alkoxycarbonyl amino acids and theirimplication in carbodiimide-mediated reactions in peptide synthesis”Can. J. Chem., 59:384. In considering the reaction above for forming anactivated ester of a carboxylic acid, e.g., an N-hydroxysuccinimideester, for example, if less than a stoichiometric amount ofN-hydroxysuccinimide is used in the activation process, one would expectthe dehydrating action of the carbodiimide to cause formation of theazlactone by the mechanism above. Additionally, since anN-hydroxysuccinimide ester is an activated form of the correspondingcarboxylic acid, the azlactone can also form from the active ester via amechanism similar to that for formation of the azlactone from the acid.Avoidance of azlactone formation, then, in forming desired products ingood yields, and corresponding compositions substantially absentimpurities such as substantially absent azlactone-containing impurities,is one nonlimiting aim of the conjugates, compositions and methodsprovided herein.

According to one or more embodiments of a multi-armed polymer prodrug inwhich drug is covalently attached to a multi-arm polymer via a glycinelinker, as described in U.S. Patent Application Publication No.2005/0112088, glycine is first conjugated to a drug containing a hydroxygroup. Then, the glycine-derivatized drug is conjugated to an active(e.g., NHS) ester of carboxymethyl PEG (PEG CM), as illustrated in thescheme below.

Based upon the foregoing discussion, it can be seen that azlactoneformation can occur during the step where a t-BOC-protected glycine isreacted with the drug alcohol. This is illustrated in the scheme below.Azlactone formation, even if formed in relatively minor amounts, canadversely impact the efficiency of that process and, at the same timeproduce a reactive impurity.

The foregoing discussion is relevant to the instant disclosure. Duringconjugation of various glycine-conjugated drug alcohol molecules, e.g.,a camptothecin with a 4-arm-PEG-CM active ester, it was recognized thatsubstitution of the end groups of the four-armed PEG polymer wassuboptimal. Such substitution values (and less than optimal drugloading) although reproducible, resulted in a product which, on average,possessed fewer than all four polymer arms having drug conjugatedthereto (i.e., a lower than ideal degree of substitution of drug uponthe four-arm polymer). Ideally, a multi-arm polymer such as describedherein will, on average, possess drug substituted on each of the polymerarms, so that the multi-arm polymer scaffold is “fully loaded”—tothereby take full advantage of the multi-arm feature of the polymer.See, e.g., Examples 3-5 in comparison to Examples 2, 6, 7, 8, 9, 10, and12.

Substitution of a given polymer arm of a multi-arm polymer may resultfrom a drug moiety never having become covalently attached to thepolymer arm. In addition, substitution of a given polymer arm of amulti-arm polymer may result from loss of the drug moiety followingester formation. In this regard, a drug moiety, e.g. a camptothecinalcohol, acts as a leaving group and, in a way similar to the reactionshown with N-hydroxysuccinimide ester, is removed. An azlactone islikely to be an intermediate owing to the likely participation of theneighboring urethane group as illustrated below.

While N-hydroxysuccinimide is generally considered to be a much betterleaving group than an alcohol (such as that present in, e.g., irinotecanor camptothecin), steric hindrance may be a driving force for thisprocess in molecules that have a tertiary alcohol (e.g., irinotecan orcamptotecin). The result, in the illustrated example, is that theterminating group is an azlactone or its hydrolysis product—theglycine-terminated PEG arm.

An azlactone, if formed, represents an impurity since it may react withamine groups of endogenous proteins to form unwanted conjugates in vivo.See U.S. Pat. No. 5,321,095.

It is believed that the conjugates, reagents, and methods describedherein possess, among other things, the advantages of increased drugloading while avoiding impurities. In addition, the instant conjugatesdemonstate superior in-vivo anti-cancer activity, and reduced toxicity,along with an efficacy that is significantly enhanced over unconjuateddrug alone.

The features of such conjugates will now be discussed in greater detailbelow.

Structural Features of the Polymer Conjugate

As described above, a conjugate as provided herein comprises a multi-armpolymer, i.e., having three or more arms, where the conjugate possessesthe following generalized structure,

where: R is an organic core radical comprising from about 3 to about 150carbon atoms; Q is a linker; POLY₁ is a water-soluble and non-peptidicpolymer segment; X is a spacer that is optionally present; R₁, in eachoccurrence, is independently selected from the group consisting of H,lower alkyl, and an electron withdrawing group; n is an integer from 1to 7; D is a residue of a small molecule, preferably a small moleculedrug, having a molecular weight of less than about 800 daltons; and q is3 or greater. Each arm of the multi-armed polymer is independent fromthe other. That is to say, each of the “q” arms of the multi-armedpolymer may be composed of a different Q, POLY₁, X, R₁, n and so forth.Typical of such embodiments, a generalized structure corresponds to:R[(-Q₁-POLY_(1A)-X₁—(CH₂)₂(CR_(1A)H)_(n1)C(O)—O-D₁)(-Q₂-POLY_(1B)-X₂—(CH₂)₂(CR_(1B)H)_(n2)C(O)—O-D₂)(-Q₃-POLY_(1C)-X₃—(CH₂)₂(CR_(A)H)_(n2)C(O)—O-D₃). . . ], and so forth for each of the arms emanating from the centralorganic core radical. Generally, however, each arm of the multi-armedprodrug is the same, with certain exceptions to be addressed in greaterdetail below.

As described in the preceding section, the multi-armed polymer containsthe feature of an alkanoate segment, an alkanoate segment within the—X—(CH₂)₂(CR₁H)_(n)C(O)—O-D moiety. The alkanoate is of a length andnature sufficient to prevent or reduce neighboring group participation(e.g., from the linker group, “X”). An alpha or beta alkyl group or thelike (e.g., when R₁ is alkyl) can offer value in providing sterichindrance to improve the selectivity of the ultimate active esterreagent. As can be seen in the supporting examples, use of the alkanoatelinker is effective to result in exemplary multi-arm polymer drugconjugates having high drug loading efficiencies.

Each of the variable components of Compound I will now be described indetail. Each of the components described herein in relationship to thecorresponding conjugate similarly extends to the correspondingmulti-armed polymer reagent, e.g., where D as presented in Compound I asa residue of a small molecule, D can be substituted for, for example, Hor a functional group corresponding to an activated ester.

Organic Core, “R”

In Compound I, R is an organic core radical possessing from about 3 toabout 150 carbon atoms. Preferably, R contains from about 3 to about 50carbon atoms, and even more preferably, R contains from about 3 to about10 carbon atoms. That is to say, R, in one embodiment, may possess anumber of carbon atoms selected from the group consisting of 3, 4, 5, 6,7, 8, 9, and 10. The organic core may optionally contain one or moreheteroatoms (e.g., 0, S, or N), depending of course on the particularcore molecule employed. R may be linear or cyclic, and typically,emanating therefrom are at least 3 independent polymer arms, at leastone of which possesses an active agent moiety covalently attachedthereto. Preferred organic core molecules are saturated aliphatics.Looking at Compound I, “q” corresponds to the number of polymer armsemanating from “R.” In some instances one or more of the polymer armsmay not have an active agent covalently attached thereto, but rather mayhave a relatively unreactive or unreacted functional group at itsterminus, typically resulting from a synthesis that has failed to go tocompletion or due to hydrolysis. In this instance, D is absent and theindividual structure of at least one of the polymer arms is in itsprecursor form (or is a derivative thereof), i.e., having at itsterminus not an active agent, D, but rather, a functional group.However, one particularly advantageous feature of the multi-armedconjugates provided herein is their degree of drug loading on each ofthe water-soluble polymer arms of the multi-armed polymer. Preferably,due to the absence of any potential neighboring group interactions, thedegree of drug loading of the multi-armed polymer (within a compositionof multi-armed polymers) is greater than about 92%. For example, for amulti-armed polymer conjugate having q polymer arms, the average drugloading (within a composition of multi-armed polymers) will preferablybe 0.92(q) or greater. Even more preferably, a multi-armed polymerconjugate as provided herein will possess a degree of drug loading(within a composition of multi-armed polymers) of at least 93%, 94%,95%, 96%, 97%, 98% or even 99% or greater.

The central core organic radical, R, corresponds to a discrete moleculethat provides a number of polymer attachment sites approximately equalto the desired number of water-soluble and non-peptidic polymer arms.Preferably, the central core molecule of the multi-arm polymer structureis the residue of a polyol, polythiol, or a polyamine bearing at leastthree hydroxyl, thiol, or amino groups available for polymer attachment.A “polyol” is a molecule comprising a plurality (3 or more) of availablehydroxyl groups. A “polythiol” is a molecule that possesses a plurality(3 or more) thiol groups. A “polyamine” is a molecule comprising aplurality (3 or more) available amino groups. Depending on the desirednumber of polymer arms, the precursor polyol, polyamine or polythiol,(prior to covalent attachment of POLY)) will typically contain 3 toabout 25 hydroxyl, or amino groups or thiol groups, respectively,preferably from 3 to about 10 hydroxyl, amino groups or thiol groups,(i.e., 3, 4, 5, 6, 7, 8, 9, 10), most preferably, will contain from 3 toabout 8 (e.g., 3, 4, 5, 6, 7, or 8) hydroxyl, amino groups or thiolgroups suitable for covalent attachment of POLY₁. The polyol, polyamineor polythiol may also include other protected or unprotected functionalgroups. Focusing on organic cores derived from polyols or polyamines,although the number of intervening atoms between each hydroxyl or aminogroup will vary, preferred cores are those having a length of from about1 to about 20 intervening core atoms, such as carbon atoms, between eachhydroxyl or amino group, preferably from about 1 to about 5. Inreferring to intervening core atoms and lengths, —CH₂—, for example, isconsidered as having a length of one intervening atom, although themethylene group itself contains three atoms total, since the Hs aresubstituents on the carbon, and —CH₂CH₂—, for instance, is considered ashaving a length of two carbon atoms, etc. The particular polyol orpolyamine precursor depends on the desired number of polymer arms in thefinal conjugate. For example, a polyol or polyamine core molecule having4 functional groups, Q, is suitable for preparing a prodrug inaccordance with Compound I having four polymer arms extending therefromand covalently attached to active agent.

The precursor polyol or polyamine core will typically possess astructure R—(OH)_(p) or R—(NH₂)_(p) prior to functionalization with apolymer. The value of p corresponds to the value of q in Compound I,since each functional group, typically —OH or —NH₂, in the parent coreorganic molecule, if sterically accessible and reactive, is covalentlyattached to a polymer arm, POLY₁. Note that in Compound I, the variable“Q,” when taken together with R, typically represents a residue of thecore organic radical as described herein. That is to say, whendescribing preferred organic core molecules, particularly by name, thecore molecules are described in their precursor form, rather than intheir radical form after removal of, for example, a proton. So, if forexample, the organic core radical is derived from pentaerythritol, theprecursor polyol possesses the structure C(CH₂OH)₄, and the organic coreradical, together with Q, corresponds to C(CH₂O—)₄, where Q is O. Thecore is non-crosslinked, and when taken together with POLY₁ excludesstar-type polymers.

Illustrative polyols that are preferred for use as the polymer coreinclude aliphatic polyols having from 1 to 10 carbon atoms and from 3 to10 hydroxyl groups, including for example, trihydroxyalkanes,tetrahydroxyalkanes, polyhydroxy alkyl ethers, polyhydroxyalkylpolyethers, and the like. Cycloaliphatic polyols include straightchained or closed-ring sugars and sugar alcohols, such as mannitol,sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol,erythritol, adonitol, dulcitol, facose, ribose, arabinose, xylose,lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose,pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose,maltose, and the like. Additional examples of aliphatic polyols includederivatives of glucose, ribose, mannose, galactose, and relatedstereoisomers. Aromatic polyols may also be used, such as1,1,1-tris(4′-hydroxyphenyl) alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, 2,6-bis(hydroxyalkyl)cresols, and thelike. Other core polyols that may be used include polyhydroxycrownethers, cyclodextrins, dextrins and other carbohydrates (e.g.,monosaccharides, oligosaccharides, and polysaccharides, starches andamylase).

Preferred polyols include glycerol, trimethylolpropane, pentaerythritol,dipentaerythritol, tripentaerythritol, ethoxylated forms of glycerol,trimethylolpropane, pentaerythritol, dipentaerythritol,tripentaerythritol. Also, preferred are reducing sugars such as sorbitoland glycerol oligomers, such as diglycerol, triglycerol, hexaglyceroland the like. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.Additionally, a polyglycerol having an average of 24 hydroxyl groups iscommercially available.

Exemplary polyamines include aliphatic polyamines such as diethylenetriamine, N,N′,N″-trimethyldiethylene triamine, pentamethyl diethylenetriamine, triethylene tetramine, tetraethylene pentamine, pentaethylenehexamine, dipropylene triamine, tripropylene tetramine,bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, andN,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines thatcan be used in the present invention include putrescine, spermidine, andspermine. Numerous suitable pentamines, tetramines, oligoamines, andpentamidine analogs suitable for use in the present invention aredescribed in Bacchi et al. (2002) Antimicrobial Agents and Chemotherapy,46(1):55-61, which is incorporated by reference herein.

Provided below are illustrative structures corresponding to the organicradical portion of the conjugate, R, and the corresponding idealizedconjugate, assuming that each of the hydroxyls in the parent polyol hasbeen transformed to a polymer arm and that each polymer arm has drugcovalently attached thereto. Note that the organic radicals shown below,derived from polyols, include the oxygens, which, in the context ofCompound I, for the arms that are polymer arms, are considered as Q. Itis not necessary that all hydroxyls in, for example, a polyol-derivedorganic radical, form part of a polymer arm. In the illustrativeexamples below, Q is shown as 0, but can equally be considered ascorresponding to S, —NH—, or —NH—C(O)—. Additionally, the last twoexemplary core structures illustrate two-polyol cores interconnected bya bifunctional linker such as the representative disulfide or dipeptideshown. Similar structures can be envisioned using any of theillustrative core molecules. See, e.g., International Patent PublicationNo. WO 2007/098466 for examples of additional multi-armed polymerssuitable for preparing the multi-armed polymer alkanoate conjugatesdescribed herein, the contents of which is expressly incorporated hereinby reference.

Organic Radical*/Illustrative Conjugate (*Includes Q)

Additional multi-armed polymer starting materials suitable for preparinga multi-armed polymer alkanoate conjugate as described herein(corresponding to R-(Q-POLY₁˜)_(q) are available from, for example, NOFCorporation (Japan) through their catalogs, which are incorporatedherein by reference.

Alternatively, a multi-armed polymer regent for preparing a multi-armedpolymer drug may be synthetically prepared. For instance, any of anumber of suitable polyol core materials can be purchased from achemical supplier such as Aldrich (St. Louis, Mo.). Certain highlyderivatized polyols are also available, e.g., a polyglycerol having anaverage of 24 hydroxyl groups, from Hyperpolymers GmbH. The terminalhydroxyls of the polyol are first converted to their anionic form,using, for example, a strong base, to provide a site suitable forinitiating polymerization, followed by direct polymerization of monomersubunits, e.g., ethylene oxide, onto the core. Chain building is allowedto continue until a desired length of polymer chain is reached in eachof the arms, followed by terminating the reaction, e.g., by quenching.

In yet another approach, an activated multi-armed polymer reagent can besynthetically prepared by first providing a desired polyol corematerial, and reacting the polyol under suitable conditions with aheterobifunctional PEG mesylate of a desired length, where thenon-mesylate PEG terminus is optionally protected to prevent reactionwith the polyol core. The resulting multi-armed polymer is then suitablefor additional transformations or direct coupling to an active agent,following deprotection if necessary.

Multi-armed polymer reagents based on polyamino cores can be prepared,for example, by direct coupling to a polymer reagent activated with anacylating agent such as an NHS ester, a succinimidyl carbonate, a BTCester or the like, to provide multi-armed polymer precursors having anamide linker, Q. Alternatively, a core molecule having multiple aminogroups can be coupled with an aldehyde terminated polymer, such as aPEG, by reductive amination (using, for example, a reducing agent suchas sodium cyanoborohydride) to provide a multi-armed polymer precursorhaving an internal amine linker, Q.

An illustrative synthesis of a dipeptide-linked pentaerythritolylcore-containing multi-armed polymer is provided herein as FIGS. 1A-1C.

Although the polymer, PEG, is described as a representative polymer inthe synthetic descriptions above, such approaches apply equally as wellto other water-soluble polymers described herein.

Linkages Q and X

The linkages between the organic radical, R, and the polymer segment,POLY₁, or between POLY₁ and the alkanoate moiety, result from thereaction of various reactive groups contained within R and POLY₁.Illustrative linking chemistry useful for preparing the polymerconjugates of the invention can be found, for example, in Wong, S. H.,(1991), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press,Boca Raton, Fla. and in Brinkley, M. (1992) “A Brief Survey of Methodsfor Preparing Protein Conjugates with Dyes, Haptens, and CrosslinkingReagents”, in Bioconjug. Chem., 3, 2013. The alkanoate portion of themulti-armed polymer conjugate provides a hydrolytically degradable bond(i.e., an ester linkage) to the small molecule active agent, so that theactive agent is released over time from the multi-armed polymer core.

The multi-arm polymeric conjugates provided herein (as well as thecorresponding reactive polymer precursor molecules, and so forth)comprise a linker segment, Q, and optionally, a spacer segment, X.Exemplary spacers or linkers can include segments such as thoseindependently selected from the group consisting of —O—, —S—, —NH—,—C(O)—, —O—C(O)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—,—C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—,—CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—.

In any of the above examples, a simple cycloalkylene group, e.g. 1,3- or1,4-cyclohexylene, may replace any two, three or four carbon alkylenegroup. For purposes of the present disclosure, however, a series ofatoms is not a spacer moiety when the series of atoms is immediatelyadjacent to a water-soluble polymer segment and the series of atoms isbut another monomer, such that the proposed spacer moiety wouldrepresent a mere extension of the polymer chain. A spacer or linker asdescribed herein may also comprise a combination of any two or more ofthe above groups, in any orientation.

Referring to Compound I, Q is a linker, preferably one that ishydrolytically stable. Typically, Q contains at least one heteratom suchas O, or S, or NH, where the atom proximal to R in Q, when takentogether with R, typically represents a residue of the core organicradical R. Generally, Q contains from 1 to about 10 atoms, or from 1 toabout 5 atoms. Q typically contains one of the following numbers ofatoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Illustrative and preferred Qsinclude 0, S, —NH—, and —NH—C(O)—. Preferably, Q is oxygen, meaning thatthe organic core molecule is a polyol.

Again in reference to Compound I, X is a spacer that connects POLY₁ withthe dimethylene group of the alkanoate segment. Generally speaking, thespacer has an atom length of from about 1 atom to about 50 atoms, ormore preferably from about 1 atoms to about 25 atoms, or even morepreferably from about 1 atom to about 10 atoms. Typically, the spacer isof an atom length selected from the group consisting of 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. When considering atom chain length, only atomscontributing to the overall distance are considered. For example, aspacer having the structure, —CH₂—C(O)—NH—CH₂ CH₂ O—CH₂ CH₂ O—C(O)—O—has a chain length of 11 atoms, since substituents are not considered tocontribute significantly to the length of the spacer. Spacer X may behydrolytically stable or hydrolytically degradable. In a particularembodiment, e.g., when POLY₁ is a polyethylene glycol, e.g., in severalof the exemplary alkanoate conjugates and corresponding multi-armpolymer reagents provided in the accompanying Examples, X is oxygen. Forinstance, when POLY₁ corresponds to the structure —(CH₂CH₂O)_(n)CH₂CH₂—,X may correspond to oxygen or —O—, such that the final structureincluding the “X’ oxygen, may, for simplification, appear in shorthandfashion as —(CH₂CH₂O)_(n)—.

In yet another embodiment, X possesses the structure: Y—Z, where Y is aspacer fragment covalently attached to Z, a hydrolytically degradablelinkage. In certain embodiments, Z itself may not constitute ahydrolytically degradable linkage, however, when taken together with Y,or at least a portion of Y, forms a linkage that is hydrolyticallydegradable.

In yet a more particular embodiment of the spacer, X, Y has thestructure: —(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂O)_(c)—,wherein each R_(x) and R_(y), in each occurrence, is independently H oran organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, and substituted aryl, a ranges from 0 to 12 (i.e., can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), b ranges from 0 to 12(i.e., can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), K isselected from —C(O)—, —C(O)NH—, —NH—C(O)—, —O—, —S—, O—C(O)—, C(O)—O—,O—C(O)—O—, O—C(O)—NH—, NH—C(O)—O—, c ranges from 0 to 25, and Z isselected from C(O)—O—, O—C(O)—O—, —O—C(O)—NH—, and NH—C(O)—O—. Theparticular structure of K and of Z will depend upon the values of eachof a, b, and c, such that none of the following linkages result in theoverall structure of spacer X: —O—O—, NH—O—, NH—NH—.

In yet another embodiment of the spacer, X, Y has the structure:—(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂NH)_(c)—, where thevariables have the values previously described. In certain instances,the presence of the short ethylene oxide or ethyl amino fragments inspacer, X, can be useful in achieving good yields during preparation ofthe prodrug conjugate, since the presence of the linker can help tocircumvent problems associated with steric hindrance, due to themulti-armed reactive polymer, the structure of the active agent, or acombination of both. Preferably, c is selected from the group consistingof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, “a” ranges from 0 to 5, i.e., is selectedfrom 0, 1, 2, 3, 4, or 5. In yet another embodiment, b ranges from 0 to5, i.e., is selected from 0, 1, 2, 3, 4, or 5. In yet anotherembodiment, c ranges from 0 to 10. In yet another embodiment, K is—C(O)—NH. Any of the embodiments described herein is meant to apply notonly to generalized Compound I, but also extend to particularcombinations of embodiments.

Spacer X, when present, may also correspond to an amino acid, di- ortri-acid or the like, or a peptide or oligopeptide, in particular due tothe presence of the alkanoate group which is sufficient to prevent anyneighboring group interactions from occurring. Suitable amino acidsinclude amino acids such as alanine, valine, leucine, isoleucine,glycine, threonine, serine, cysteine, methionine, tyrosine,phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine,arginine, histidine, proline, and the like, as well as non-naturallyoccurring amino acids.

A preferred spacer, when present, is oxygen (—O—).

The Polymer, POLY1

In Compound I, POLY₁ represents a water-soluble and non-peptidicpolymer. POLY₁ in each polymer arm of Compound I is independentlyselected, although preferably, each polymer arm will comprise the samepolymer. That is to say, most preferably, each POLY₁ in each arm of themulti-armed polymer conjugate is the same. Preferably, each of the arms,i.e., each “(-Q-POLY₁-X-D)” of Compound I is also identical. Any of avariety of polymers that are non-peptidic and water-soluble can be usedto form a conjugate in accordance with the present invention. Examplesof suitable polymers include, but are not limited to, poly(alkyleneglycols), copolymers of ethylene glycol and propylene glycol,poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures of any one or more of the above.

Preferably, POLY₁ is a polyethylene glycol or PEG. POLY₁ can be in anyof a number of geometries or forms, including linear chains, branched,forked, etc., although preferably POLY₁ is linear (i.e., in each arm ofthe overall multi-arm structure) or forked. A preferred structure for amulti-armed polymer prodrug having a “forked” polymer configuration isas follows:

F represents a forking group, and the remaining variables are aspreviously described. Preferably, the fork point in the forking group,F, comprises or is (—CH), although it may also be a nitrogen atom (N).In this way, each polymer arm is forked to possess two active agentmoieties releasably covalently attached thereto, rather than one.

Illustrative forked polymers useful for preparing a multi-armed polymerof the type shown in structure XII are described in U.S. Pat. No.6,362,254.

When POLY₁ is PEG, its structure typically comprises —(CH₂CH₂O)_(n)—,which may also be represented as —(CH₂CH₂O)_(n)CH₂CH₂—, where n mayrange from about 5 to about 400, preferably from about 10 to about 350,or from about 20 to about 300. In a preferred embodiment of themulti-armed polymer conjugates provided herein, POLY₁ is linearpolyethylene glycol.

In the multi-arm embodiments described here, each polymer arm, POLY₁,typically has a molecular weight corresponding to one of the following:200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500,18,000, 19,000, 20,000 daltons or greater. Overall molecular weights forthe multi-armed polymer configurations described herein (that is to say,the molecular weight of the multi-armed polymer as a whole) generallycorrespond to one of the following: 800, 1000, 1200, 1600, 2000, 2400,2800, 3200, 3600, 4000, 5000, 6000, 8000, 10,000, 12,000, 15,000,16,000, 20,000, 24,000, 25,000, 28,000, 30,000, 32,000, 36,000, 40,000,45,000, 48,000, 50,000, 60,000, 80,000 or 100,000 or greater.

Typically, the overall molecular weight for a multi-armed polymer of theinvention ranges from about 800 to about 80,000 daltons, or from about900 to about 70,000 daltons. Other preferred molecular weight ranges fora multi-armed polymer of the invention are from about 1,000 to about40,000 daltons, or from about 5,000 to about 30,000 daltons, or evenfrom about 20,000 to about 80,000 daltons for higher molecular weightembodiments of the present conjugates.

The Alkanoate Segment

The multi-armed polymer conjugates provided herein comprise, in qpolymer arms, an alkanoate segment. The alkanoate segment is provided toboth conjugate to and then ultimately release the drug. Also, thealkanoate structure is designed to prevent the occurrence of possibleneighboring group interactions that can lead to less than optimal drugloading and the formation of undesirable impurities in multi-armedpolymer conjugate compositions. This is provided by both the length ofthe alkanoate segment and by the particular atoms and functional groupscontained therein. Each of q polymer arms as illustrated in Compound Iterminates in an alkanoate segment, in which the small molecule drug (or-hydrogen or active ester functionality) is covalently attached to thepolymer arm via an ester linkage (forming part of the alkanoate). Thealkanoate segment corresponds to: —(CH₂)₂—(CHR₁)_(n)C(O)—O-D, where R₁is, in each occurrence, independently H, lower alkyl, alkylene, or anelectron withdrawing group, and n is an integer from 1 to about 7. Ascan be seen from R₁, the carbon in the position alpha, beta, gamma,etc., to the carbonyl may be independently substituted, e.g., with alower alkyl, alkylene, or electron withdrawing group. A substituent,such as a methyl or like group, in the alpha position, and to a lesserextent in the beta or gamma position, can provide steric hindrance toallow greater control or selectivity of the reagent, such that itsreactivity with nucleophilic groups such as those present on drugmolecules (e.g., alcohols, thiols) is diminished. Such a substituent mayprovide additional control of the hydrolytic stability of multi-armedconjugate. See, e.g., U.S. Pat. No. 6,495,659, the contents of which areincorporated herein by reference. Typically, the presence of an alkylgroup at the alpha position will impart additional hydrolytic stabilityto the adjacent ester bond over that observed for such alkanoate absentsuch alkyl group.

R₁ may be, for example, H, or an alkyl group containing from 1 to about6 carbon atoms. The lower alkyl group may be straight chain or branched,as exemplified by methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, i-pentyl, and the like. If an alkylene groupis substituted on, e.g., the alpha position, and tied back to, e.g., thedelta position, the structure has an alkanoate group that is cyclic innature, as shown below.

Alternatively, R₁ may be an electron withdrawing group (EWG) such as ahalide (e.g., F, Cl, Br, or I), a nitrile, —NO₂, CF₃, —SO₃, or any otherEWG commonly known in the art. The integer, n, typically has a valueselected from 1, 2, 3, 4, 5, 6, and 7.

Thus, the portion —(CHR₁)_(n)— may correspond to any one of —CHR₁—,—CHR₁—CHR₁—, —CHR₁—CHR₁—CHR₁, —CHR₁—CHR₁—CHR₁—CHR₁,—CHR₁—CHR₁—CHR₁—CHR₁—CHR₁, —CHR₁CHR₁—CHR₁—CHR₁—CHR₁—CHR₁,—CHR₁—CHR₁—CHR₁—CHR₁—CHR₁—CHR₁—CHR₁, where each R₁ in each chain isindependently selected. In one particular embodiment, each R₁ in each ofthe foregoing, is hydrogen. In yet another embodiment, in each of theforegoing, each R₁ is hydrogen with the exception of the R₁ in theposition alpha or adjacent to the carbonyl, in which case, thisparticular R₁ alone is lower alkyl. In yet an additional embodiment, ineach of the foregoing structures, each R₁ is hydrogen with the exceptionof the R₁ in the position alpha or adjacent to the carbonyl, in whichcase, this particular R₁ alone is an EWG as described above.

Active Agent, D

Returning now to Compound I, D represents the residue of a smallmolecule active agent, and q (the number of polymer arms) ranges fromabout 3 to about 50. Illustrative ranges are from about 3 to about 10,from about 11 to about 25, from about 26 to 40, or from about 41 toabout 50. Preferably, q ranges from about 3 to about 25. Morepreferably, q is from 3 to about 10, e.g., q possesses a value of 3, 4,5, 6, 7, 8, 9, or 10. In a preferred embodiment, q is four. The activeagent residue, D contains at least one hydroxyl functional groupsuitable for covalent attachment to the multi-armed polymer describedherein to form a hydrolyzable ester linkage, such that upon hydrolysis,the active agent is released in its unmodified form.

In accordance with one embodiment of the invention, a multi-armedpolymer conjugate is characterized as having from about 3 to about 25active agent molecules covalently attached thereto. More particularly,the conjugate possesses 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 active agent molecules covalentlyattached thereto. In a further embodiment, the conjugate of theinvention has from about 3 to about 8 active agent molecules covalentlyattached to the water-soluble polymer. Typically, although notnecessarily, the number of polymer arms will correspond to the number ofactive agents covalently attached to the water-soluble polymer. That isto say, in the case of a polymer having a certain number of polymer arms(e.g, q), each having a reactive alkanoate functional group at itsterminus, the number of active agents covalently attached thereto in theresulting conjugate is most desirably q. In a preferred embodiment, acomposition comprising the subject multi-armed polymer conjugate ischaracterized by a degree of drug loading of 92 percent or greater. Thatis to say, for a composition containing a multi-armed polymer conjugatehaving q polymer arms, the composition is characterized as having a drugloading on average per species of 0.92(q) or greater. That is to say,the composition is characterized by a drug loading on average perspecies satisfying or more of the following: 0.92(q) or greater; 0.93(q)or greater; 0.94(q) or greater; 0.95(q) or greater; 0.96(q) or greater;0.97(q) or greater; 0.98(q) or greater; 0.99(q) or greater; and 1(q).

In yet another embodiment, rather than having multiple polymer armsemanating from a central organic radical core, a conjugate of theinvention is characterized as a water-soluble polymer having pendantactive agent moieties covalently attached thereto, each preferablycovalently attached by a degradable linkage such as the alkanoatelinkage described herein. In such an embodiment, the structure of thepolymer prodrug conjugate is described generally asPOLY₁(-X—(CH₂)₂(CR₁H)_(n)C(O)—O-D)_(q), where each drug residue iscovalently attached to POLY₁ via the alkanoate linker, and the variablesPOLY₁, X, R₁, n, D, and q are as set forth above, and the polymer,typically a linear polymer, possesses “q” active agent residues attachedthereto, typically at discrete lengths along the polymer chain, viaspacer X, which is connected to the alkanoate linker-drug moiety.

In a specific embodiment, the active agent moiety or residue is a smallmolecule possessing a molecular weight of less than about 1000. In yetadditional embodiments, the small molecule drug possesses a molecularweight of less than about 800, or even less than about 750. In yetanother embodiment, the small molecule drug possesses a molecular weightof less than about 500 or, in some instances, even less than about 300.

Preferred active agent moieties are anticancer agents. Particularlypreferred are oncolytics bearing at least one hydroxyl group (i.e.,suitable for forming the alkanoate attachment). Particularly preferredare alkaloid cytotoxic agents such as the taxanes and camptothecins, aswell as the vinca alkaloids vincristine, vinorelbine, vinblastine, andviddesine.

One preferred class of active agents is the camptothecins. In apreferred embodiment, D is a taxane or taxane derivative such aspaclitaxel or docetaxel. For purposes of the present disclosure, theterm “taxane” includes all compounds within the taxane family ofterpenes. Thus, taxol (paclitaxel), 3′-substitutedtert-butoxy-carbonyl-amine derivatives (taxoteres) and the like as wellas other analogs available from, for example, Sigma-Aldrich, are withinthe scope of the present disclosure. One particularly preferred D isdocetaxel, where the H at the 2′ position is absent in the finalmulti-armed polymer conjugate:

Other preferred taxanes are those capable of undergoing modification atthe 2′ position of the taxane skeleton, such as described herein incertain embodiments and in the accompanying examples.

Additionally, within the scope of the disclosure are taxanes such asdocetaxel, where attachment to the multi-armed alkanoate scaffold may beat any —OH position within the taxane skeleton.

Yet another preferred class of active agents is the camptothecins. Inone embodiment, a camptothecin for use in the invention corresponds tothe structure,

wherein:

R₁-R₅ are each independently selected from the group consisting ofhydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy;

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and

L is the site of attachment to the multi-armed polymer conjugate.

Although L is shown at the 20 ring position above, the site ofattachment may be at any suitable position within the camptothecinstructure.

The term “camptothecin compound” as used herein includes the plantalkaloid 20(S)-camptothecin, as well as pharmaceutically activederivatives, analogues and metabolites thereof. Examples of camptothecinderivatives include, but are not limited to, 9-nitro-20(S)-camptothecin,9-amino-20(S)-camptothecin, 9-methyl-camptothecin,9-chloro-camptothecin, 9-flouro-camptothecin, 7-ethyl camptothecin,10-methyl-camptothecin, 10-chloro-camptothecin, 10-bromo-camptothecin,10-fluoro-camptothecin, 9-methoxy-camptothecin, 11-fluoro-camptothecin,7-ethyl-10-hydroxy camptothecin (SN38), 10,11-methylenedioxycamptothecin, and 10,11-ethylenedioxy camptothecin, and7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin,7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothecin,9-hydroxy-camptothecin, and 11-hydroxy-camptothecin. Particularlypreferred camptothecin compounds include camptothecin, irinotecan, andtopotecan.

Native and unsubstituted, the plant alkaloid camptothecin can beobtained by purification of the natural extract, or may be obtained fromthe Stehlin Foundation for Cancer Research (Houston, Tex.). Substitutedcamptothecins can be obtained using methods known in the literature orcan be obtained from commercial suppliers. For example,9-nitro-camptothecin may be obtained from SuperGen, Inc. (San Ramon,Calif.), and 9-amino-camptothecin may be obtained from IdecPharmaceuticals (San Diego, Calif.). Camptothecin and various analoguesand derivatives may also be obtained from standard fine chemical supplyhouses, such as Sigma Chemicals.

Certain preferred camptothecin compounds correspond to the generalizedstructure below

wherein:

R₁-R₅ are each independently selected from the group consisting ofhydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; and

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl.

Exemplary substituting groups include hydroxyl, amino, substitutedamino, halo, alkoxy, alkyl, cyano, nitro, hydroxycarbonyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonylamino, aryl (e.g.,phenyl), heterocycle, and glycosyl groups.

For example, in embodiment, D is irinotecan, where the H on the20-position hydroxyl is absent in the final multi-armed prodrugconjugate.

Alternatively, D is SN-38.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules,oligopeptides, polypeptides or protein mimetics, fragments, oranalogues, steroids, nucleotides, oligonucleotides, electrolytes, andthe like, and typically contains as least one free hydroxyl group, orthe like (i.e., “handle”) suitable for covalent attachment to themulti-armed polymer.

Alternatively, the drug is modified by introduction of a suitable“handle,” preferably by conversion of one of its existing functionalgroups to a functional group suitable for formation of the hereindescribed alkanoate linkage. Ideally, such a modification should notadversely impact the therapeutic effect or activity of the active agentto a significant degree. That is to say, any modification of an activeagent to facilitate its attachment to a multi-armed polymer of theinvention should result in no greater than about a 30% reduction of itsbioactivity relative to the known parent active agent prior tomodification. More preferably, any modification of an active agent tofacilitate its attachment to a multi-armed polymer of the inventionpreferably results in a reduction of its activity relative to the knownparent active agent prior to modification of no greater than about 25%,20%, 15%, 10% or 5%.

The above exemplary drugs are meant to encompass, where applicable,analogues, agonists, antagonists, inhibitors, isomers, polymorphs, andpharmaceutically acceptable salt forms thereof

Compositions/Populations of Prodrug Conjugates

As stated above, in certain instances, a composition comprising amulti-arm polymer conjugate as described herein may contain a species ofthe prodrug having one of more of its polymer arms absent drug, D. Suchan occurrence may arise, for example, due to incomplete reaction of themulti-armed reactive polymer with drug, D. Often, even in the instanceof favorable stoichiometry, i.e., using an excess of drug relative tothe number of reactive polymer arms, it can be difficult to drive thereaction to completion such that the product may comprise a mixturepolymer species. However, by way of employing the present alkanoatelinker, when characterizing a multi-armed polymer prodrug composition asprovided herein, the degree (i.e., percentage) of drug loading for thefinal conjugate is advantageously high, typically at 92% or greater,e.g., from about 92% to 100% loading.

However, for the sake of completeness, it should be noted that acomposition of the invention may, in certain instances, comprise amulti-arm polymer conjugate generally characterized asR(-Q₁-POLY₁-X₁—(CH₂)₂(CR₁H)_(n)C(O)—O-D)_(q), where one or more polymerarms are absent drug. Assuming that each arm of the starting multi-armedpolymer reagent has an alkanoic acid function attached thereto, theresulting conjugate may, and preferably will, possess drug covalentlyattached to each polymer arm, or alternatively, may lack drug, D. Thevariable D is a small molecule, where D₁ indicates the presence of D andD₀ indicates its absence. So, for example, a composition may comprise:

R(-Q-POLY₁-X—(CH₂)₂(CR₁H)_(n)C(O)—O-D)_(m)(-Q-POLY₁-X—(CH₂)₂(CR₁H)_(n)C(O)—O-D₀)_(s),

where m+s=q, the only caveat being that m (the number of polymer armshaving drug attached) is 1 or greater (e.g., from 1 to q). The value ofm will correspondingly range from 0 to (q−1). Typically, when drug isabsent, i.e., is D₀, the polymer arm will terminate in functional group,Y, where Y is —H or a derivative (reaction product) of the correspondingalkanoic acid or activated carboxysuccinimide. More specifically, acomposition of the invention may comprise one or more multi-armedpolymer conjugate species having the structure:

R(-Q-POLY₁-X—(CH₂)₂(CR₁H)_(n)C(O)—O-D)_(m)(-Q-POLY₁-X—(CH₂)₂(CR₁H)_(n)C(O)—O—Y)_(s),

where Y corresponds to H or a derivative of the corresponding alkanoicacid or activated carboxysuccinimide. Preferred compositions are thosein which, when characterized overall, possess a value of m that is0.92(q) or greater. Ideally, in the instance of quantitativesubstitution of active agent, s equals zero and m equals q.

As an illustration, in an instance in which the multi-armed polymerconjugate contains four polymer arms, the idealized value of the numberof covalently attached drug molecules per multi-armed polymer is four,and an average number of drug molecules per multi-armed polymer rangesfrom about 92% to about 100% of the idealized value. This corresponds toan average number of D per multi-arm polymer conjugate ranging fromabout 3.68 to 4.0.

In yet another embodiment, for a multi-aimed polymer conjugatecomposition, e.g., where the number of polymer arms ranges from about 3to about 8, the majority species present in the composition are thosehaving either an idealized number of drug molecules attached to thepolymer core (“q”) or those having a combination of (“q”) and (“q−1”)drug molecules attached to the polymer core.

For example, a preferred multi-armed polymer prodrug composition inaccordance with the invention may comprise one or more of the followingconjugate species:

and so forth, where O-Doc corresponds to:

and preferably predominantly comprises the first species, that is, whereeach polymer arm has drug, in this case, docetaxel, covalently attachedthereto. That is to say, most preferably, a multi-arm polymer conjugateas provided herein possesses active agent covalently attached to eachpolymer arm, such that essentially quantitative substitution of activeagent in each of the polymer arms has taken place. In such anembodiment, a composition of the invention is characterized by having anaverage number of drug molecules per multi-armed polymer thatcorresponds essentially to its idealized value (i.e., is essentially100% of its idealized value).

This disclosure is meant to encompass each prodrug species describedherein, whether described singly or as forming part of a prodrugcomposition.

One exemplary and preferred conjugate is described in Example 2,4-ARM-PEG_(20K)-BA-DOC. That is to say, in a preferred embodiment, amulti-armed polymer conjugate as provided herein possesses apentaerythritol-derived core (such that R-Q-corresponds topentaerythritolyl), a POLY₁ that corresponds to PEG,—(CH₂CH₂O)_(n)CH₂CH₂—, a value of X that corresponds to oxygen, O, andan alkanoate linker portion that corresponds to butanoate, such thatn=1, R₁═H, and q equals 4. As described in Example 2, the substitutionvalue for the four-armed polymer conjugate was approximately 98-100%.Additional characterization of extent of drug loading is provided inExample 11 (see, e.g., Table 1). As can be seen from the comparativedata provided in Example 11, average drug loading values forglycine-linked and carboxymethylene-linked multi-armed conjugates weresignificantly lower, averaging from about 75% to about 80% of theiridealized values, indicating the value of the instant alkanoate-linkedmulti-armed polymer conjugates and their improvement over earlierdescribed multi-armed polymer conjugates.

The preparation of additional exemplary multi-armed polymer conjugateshaving an alkanoate linker is described in Example 12. Such conjugatespossess the following features: a pentaerythritolyl core (e.g., whereR-Q corresponds to pentaerythritolyl), a water-soluble and non-peptidicpolymer segment, POLY), that is PEG, —(CH₂CH₂O)_(n)CH₂CH₂—, a spacer, X,that is oxygen, a value of q that equals 4, and the following alkanoatelinkers: α-methylpropanoate, α-methylpentanoate, hexanoate, octanoate,and decanoate.

Method of Forming a Multi-Armed Polymer Prodrug Conjugate

Multi-armed reactive polymers, such as those for preparing a conjugateof the invention can be readily prepared from commercially availablestarting materials in view of the guidance presented herein, coupledwith what is known in the art of chemical synthesis.

Hydroxyl-terminated multi-armed PEGs having either a pentaerythritolcore or a glycerol core are available from NOF Corporation. Suchmulti-armed PEGs can be used directly for preparing the conjugatesprovided herein, by functionalization to prepare the correspondingalkanoic acids for coupling to hydroxyls present on an active agent.See, e.g., Example 1, which describes formation of 4-ARM-PEG-ButanoicAcid, followed by conjugation as described in Example 2. In the approachdescribed, the multi-arm 4-ARM-PEG-OH starting material is reacted witha protected bromobutanoic acid in the presence of a strong base to formthe desired multi-armed PEG alkanoic acid reagent. See, e.g., U.S.Patent Application Publication No. US 2005/0036978. This approach isapplicable to the preparation of any multi-armed PEG-OH startingmaterial. The resulting multi-armed polymer alkanoic acid can be coupledto the target hydroxyl-containing drug, e.g., using a suitablecondensing agent such as diisopropylcarbodiimide (DIC). This is shownschematically below,

In an alternative approach, the drug is functionalized to contain thealkanoic acid moiety, followed by covalent attachment to the multi-armedpolymer to provide a conjugate as described herein. This method isillustrated below using a PEG BTC active ester to ultimately yield aurethane bond between the linker and the PEG segment. Many variations onthe methods described can be envisioned by one skilled in the art.

The methods described herein are flexible enough to allow manyvariations in the exact structures of the reactants and products. Forexample, one may readily substitute alkyl chlorides or mesylates for therespective alkyl bromides in substitution reactions. Also, where activeNHS esters are used for illustration, other active esters, such asp-nitrophenolates or BTCs can be used. Also, extender groups, especiallythose containing ethyleneoxy subunits, may be used as a second linker,between the core multiarm PEG structure and the terminating linker.Selection of suitable functional groups, linkers, protecting groups, andthe like to achieve a multi-arm polymer prodrug in accordance with theinvention, will depend, in part, on the functional groups on the activeagent and on the multi-armed polymer starting material and will beapparent to one skilled in the art, based upon the contents of thepresent disclosure.

The prodrug product may be further purified. Methods of purification andisolation include precipitation followed by filtration and drying, aswell as chromatography. Suitable chromatographic methods include gelfiltration chromatography, ion exchange chromatography, and BiotageFlash chromatography.

Pharmaceutical Compositions

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or moremulti-armed polymer conjugates of the invention or a pharmaceuticallyacceptable salt thereof, with one or more pharmaceutically acceptablecarriers, and optionally any other therapeutic ingredients, stabilizers,or the like. The carrier(s) must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not unduly deleterious to the recipient thereof. The compositions ofthe invention may also include polymeric excipients/additives orcarriers, e.g., polyvinylpyrrolidones, derivatized celluloses such ashydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin. The compositions may further includediluents, buffers, binders, disintegrants, thickeners, lubricants,preservatives (including antioxidants), flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80,” andpluronics such as F68 and F88, available from BASF), sorbitan esters,lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, fatty acids and fattyesters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy,”19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference,” 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients,” Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The prodrugs of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, ophthalmic,or parenteral (including intraperitoneal, intravenous, subcutaneous, orintramuscular injection) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active agent or compound (i.e., the prodrug)into association with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by bringing theactive compound into association with a liquid carrier to form asolution or a suspension, or alternatively, bringing the active compoundinto association with formulation components suitable for forming asolid, optionally a particulate product, and then, if warranted, shapingthe product into a desired delivery form. Solid formulations of theinvention, when particulate, will typically comprise particles withsizes ranging from about 1 nanometer to about 500 microns. In general,for solid formulations intended for intravenous administration,particles will typically range from about 1 nm to about 10 microns indiameter. Particularly preferred are sterile, lyophilized compositionsthat are reconstituted in an aqueous vehicle prior to injection.

A preferred formulation is a solid formulation comprising the multi-armpolymer conjugate where the active agent, D, is docetaxel. The solidformulation is typically diluted with 5% dextrose injection or 0.9%sodium chloride injection prior to intravenous infusion.

The amount of multi-armed polymer conjugate in the formulation will varydepending upon the specific active agent employed, its activity, themolecular weight of the conjugate, and other factors such as dosageform, target patient population, and other considerations, and willgenerally be readily determined by one skilled in the art. The amount ofconjugate in the formulation will be that amount necessary to deliver atherapeutically effective amount of the compound, e.g., an alkaloidanticancer agent, to a patient in need thereof to achieve at least oneof the therapeutic effects associated with the compound, e.g., fortreatment of cancer. In practice, this will vary widely depending uponthe particular conjugate, its activity, the severity of the condition tobe treated, the patient population, the stability of the formulation,and the like. Compositions will generally contain anywhere from about 1%by weight to about 99% by weight conjugate, typically from about 2% toabout 95% by weight conjugate, and more typically from about 5% to 85%by weight conjugate, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of conjugate: 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, or more by weight.

Compositions of the present invention suitable for oral administrationmay be provided as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theconjugate as a powder or granules; or a suspension in an aqueous liquoror non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the prodrug conjugate, whichcan be formulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of themulti-armed polymer conjugate with preservative agents and isotonicagents. Such formulations are preferably adjusted to a pH and isotonicstate compatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the multi-armed polymer conjugatedissolved or suspended in one or more media such as mineral oil,petroleum, polyhydroxy alcohols or other bases used for topicalformulations. The addition of other accessory ingredients as noted abovemay be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, e.g., by inhalation. These formulationscomprise a solution or suspension of the desired multi-armed polymerconjugate or a salt thereof. The desired formulation may be placed in asmall chamber and nebulized. Nebulization may be accomplished bycompressed air or by ultrasonic energy to form a plurality of liquiddroplets or solid particles comprising the conjugates or salts thereof.

Features of the Multi-Armed Polymer Prodrug Conjugate

The pharmacokinetics of exemplary multi-armed polymer prodrugs inaccordance with the disclosure was evaluated in different animal modelsto determine, at least in part, whether sustained systemic exposure todrug was achieved upon administration. The examples provided hereindemonstrate that sustained systemic exposure to drug, e.g., docetaxel,is achieved upon administration of a multi-armed prodrug having thefeatures described herein, as supported by various in-vivo studies, andmoreover, that such increased and sustained exposure to drug iseffective to contribute to the superior antitumor activity observed forthe subject prodrugs of the instant disclosure.

As an illustration, Example 14 describes administration of4-ARM-PEG_(20K)-BA-docetaxel versus docetaxel to rats. Based upon theresults described herein, 4-ARM-PEG_(20K)-BA-docetaxel when administeredto rats exhibits a low clearance and low volume of distribution,resulting in a long 4-ARM-PEG_(20K)-BA-docetaxel terminal half life ofabout 65 hours. Plasma docetaxel C_(max) is approximately 6-fold lowerfollowing administration of equivalent 4-ARM-PEG_(20K)-BA-docetaxelcompared to docetaxel doses, while AUC is similar. Plasma docetaxelhalf-life is estimated to be about 4-fold longer following4-ARM-PEG_(20K)-BA-docetaxel administration than that observed followingdocetaxel administration (168 vs. 40 hours), thereby demonstratingsustained systemic docetaxel exposure upon administration of prodrug.

In further support of the exemplary prodrugs described herein asproviding sustained systemic exposure to drug (e.g., docetaxel) uponadministration, Example 15 demonstrates similar results in dogs.Specifically, Example 15 demonstrates a long4-ARM-PEG_(20K)-BA-docetaxel terminal half-life of 31 hours. Plasmadocetaxel C_(max) and AUC are about 110- and 7-fold lower, respectively,following administration of equivalent 4-ARM-PEG_(20K)-BA-docetaxel anddocetaxel doses. Moreover, the plasma docetaxel half-life followingadministration of 4-ARM-PEG_(20K)-BA-docetaxel is estimated to be about8-fold longer than that observed following docetaxel administration (199vs. 25 hours).

Population modeling methods applied to the pharmacokinetic data obtainedfurther support that plasma docetaxel concentrations are sustained foran extended and prolonged period following administration of4-ARM-PEG_(20K)-BA-docetaxel relative to administration of docetaxel,independent of species and dose as described in Example 16.

The observed and enhanced antitumor activity of the exemplary prodrugsof the disclosure is described in greater detail below and in theaccompanying examples.

Methods of Use

The multi-armed polymer prodrugs provided herein can be used to treat orprevent any condition responsive to the unmodified active agent in anyanimal, particularly in mammals, including humans.

The multi-arm polymer conjugates of the invention are particularlyuseful as anticancer agents, i.e., have been shown to be effective insignificantly reducing the growth of certain solid tumors as evidencedby representative in-vivo studies provided herein. That is to say, whenthe small molecule drug is an anticancer agent such as a camptothecincompound or other oncolytic such as docetaxel, the conjugates providedherein may be used to treat cancer. Types of cancer suitable fortreatment by administering a multi-armed polymer alkanoate conjugates asprovided herein include breast cancer, ovarian cancer, colon cancer,colorectal cancer, prostate cancer, gastric cancer, malignant melanoma,small cell lung cancer, non-small cell lung cancer, thyroid cancers,kidney cancer, cancer of the bile duct, brain cancer, cancer of the headand neck, lymphomas, leukemias, rhabdomyosarcoma, neuroblastoma, and thelike. The multi-arm polymer conjugates of the invention are particularlyeffective in targeting and accumulating in solid tumors. The multi-armpolymer conjugates are also useful in the treatment of HIV and otherviruses.

The multi-arm polymer conjugate may also provide improved anti-tumoractivity in patients with refractory cancer, i.e., cancer that has notresponded to various other treatments. Also, an added advantage ofadministering the multi-arm polymer conjugate is the likelihood ofreduced patient myelosuppression when compared to administration ofun-modifed active agent.

Methods of treatment comprise administering to a mammal in need thereofa therapeutically effective amount of a composition or formulationcontaining a multi-arm polymer conjugate as provided herein. Atherapeutically effective dosage amount of any specific multi-armpolymer conjugate will vary from conjugate to conjugate, patient topatient, and will depend upon factors such as the condition of thepatient, the activity of the particular active agent employed, the routeof delivery, and condition being treated.

For camptothecin-type active agents, dosages from about 0.5 to about 100mg camptothecin/kg body weight, preferably from about 10.0 to about 60mg/kg, are preferred. For taxane-type active agents, dosages from about5 to about 500 mg/m², preferably from about 25 to about 125 mg/m² arepreferred, based upon the amount of the taxane moiety. When administeredconjointly with other pharmaceutically active agents, even less of themulti-arm polymer conjugate may be therapeutically effective. The rangeset above is illustrative and those skilled in the art will determineoptimal dosing of the multi-arm polymer conjugate based on clinicalexperience and the particular treatment indication.

Methods of treatment also include administering a therapeuticallyeffective amount of a composition or formulation comprising a multi-armpolymer conjugate of an anticancer agent, e.g., a camptothecin or taxanecompound as described herein, in conjunction with a second anticanceragent. For example, in the treatment of colorectal cancer, a multi-aimpolymer prodrug of a camptothecin or docetaxel type compound may beadministered in conjuction with chemotherapeutics such as 5-fluorouracilor leucovorin xeloda, or with agents such as avastin, Erbitux®(cetuximab), or Vectibix™ (panitumumab). In the treatment of breastcancer, therapy may include administration of a multi-arm polymerconjugate as described herein, optionally in combination with xeloda,paclitaxel, docetaxel, or abraxane. In treating lung cancer, therapy mayinclude, along with administration of a multi-arm polymer conjugate ofthe invention, administration of cis-platin, carboplatin, gemcitabine,alimpta, and docetaxel (the latter in the instance in which themulti-arm polymer conjugate itself does not comprise docetaxel).

In one embodiment, a multi-armed polymer camptothecin-containingconjugate as described herein is administered in combination with5-fluorouracil and folinic acid, as described in U.S. Pat. No.6,403,569.

The multi-arm polymer conjugate of the invention may be administeredonce or several times a day, preferably once a day or less. Illustrativedosing schedules include once per week, once every two weeks, or onceevery three weeks. In the instance of a maintenance dose, dosing maytake place even less frequently than once every three weeks, such asonce monthly. The duration of the treatment may be once per day for aperiod of from two to three weeks and may continue for a period ofmonths or even years. The daily dose can be administered either by asingle dose in the form of an individual dosage unit or several smallerdosage units or by multiple administration of subdivided dosages atcertain intervals.

The multi-arm polymer conjugates provided herein exhibit improvedefficacy, improved tolerability and reduced vehicle-associated toxicitywhen compared to administration of the corresponding unmodified smallmolecule drug, absent the multi-arm polymer alkanoate scaffold.

Supporting examples illustrate the anti-tumor activity of arepresentative multi-armed polymer alkanoate conjugate in variousin-vivo mouse xenograft models for various types of cancer. As describedin detail in Example 13, 4-ARM-PEG_(20K)-BA-DOC exhibits greateranti-tumor activity than docetaxel per se in H460 and LS 174T mousexenograft models. Partial regressions were observed in two of the threecell lines examined for 4-ARM-PEG_(20K)-BA-DOC, while no regressionswere observed for docetaxel. Additionally, as shown in Example 13, atthe maximum tolerated dose, the percentage tumor growth delay (TGD) for4-ARM-PEG_(20K)-BA-DOC was 2.5-, 2.0-, and 1.6-fold—greater than fordocetaxel in 1-1460, LoVo, and LS174T xenograft models, respectively—allpointing to the vastly superior features of the multi-armed conjugatesprovided herein.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully described in theliterature. Reagents and materials are commercially available unlessspecifically stated to the contrary. See, for example, M. B. Smith andJ. March, March's Advanced Organic Chemistry: Reactions Mechanisms andStructure, 6th Ed. (New York: Wiley-Interscience, 2007), supra, andComprehensive Organic Functional Group Transformations II, Volumes 1-7,Second Ed.: A Comprehensive Review of the Synthetic Literature 1995-2003(Organic Chemistry Series), Eds. Katritsky, A. R., et al., ElsevierScience.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

The following examples illustrate certain aspects and advantages of thepresent invention, however, the present invention is in no wayconsidered to be limited to the particular embodiments described below.

Abbreviations

CM carboxymethyl or carboxymethylene (—CH₂COOH)

DCC 1,3-dicyclohexylcarbodiimide

DCM dichloromethane

DIC N,N′-diisopropylcarbodiimide

DPTS 4-(dimethylamino)-pyridinium-p-toluenesulfonate

DMF dimethylformamide

DMAP 4-(N,N-dimethylamino)pyridine

DMSO dimethyl sulfoxide

DI deionized

HCl hydrochloric acid

HOBT hydroxybenzyltriazole

HPLC high performance liquid chromatography

IPA isopropyl alcohol

K or kDa kilodaltons

MALDI-TOF Matrix Assisted Laser Desorption Ionization Time-of-Flight

MeOH methanol

MW molecular weight

NMR nuclear magnetic resonance

RT room temperature

SCM succinimidylcarboxymethyl (—CH₂—COO—N-succinimidyl)

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEC size exclusion chromatography

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

Materials and Methods

Docetaxel (Taxotere®) was purchased from Hangzhou HETD Pharm & Chem Co.,Ltd, The People's Republic of China.

Pentaerythitol based 4-ARM-PEG_(10K)-OH, 4-ARM-PEG_(20K)-OH,4-ARM-PEG_(30K)-OH, and 4-ARM-PEG_(40K)-OH were obtained from NOFCorporation (Japan). 4-ARM-PEG_(20K)-OH possesses the followingstructure: C—(CH₂O—(CH₂CH₂O)_(n)H)₄, as shown structurally below:

The variable “n” in each arm represents the number of monomer subunitscorresponding to a PEG average molecular weight of 2.5, 5, 7.5, or 10kilodaltons, such that the shorthand abbreviation for the 4-armstructure indicates an overall average molecular weight for the polymerportion of the molecule of 10, 20, 30, or 40 kilodaltons, respectively.

All ¹HNMR data was generated by a 300 or 400 MHz NMR spectrometermanufactured by Bruker.

Example 1 Synthesis of 4-ARM-PEG_(20K)-Butanoic Acid(“4-ARM-PEG_(20K)-BA”)

A solution of pentaerythritol-based 4-ARM-PEG_(20K)-OH (100.0 g, 0.020equivalents) (NOF Corporation) in toluene (750 ml) was azeotropicallydried by distilling off 150 ml of toluene. 1.0M solution of potassiumtert-butoxide in tert-butanol (60 ml, 0.060 moles) and1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (12.6 g,0.052 moles) were added and the mixture was stirred overnight at 70° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (1,000 ml).The pH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 by addition of 1M sodium hydroxide, and thesolution was stirred for two hours keeping the pH at 12 by periodicaddition of 1M sodium hydroxide. Thereafter, the pH was adjusted to 3 byaddition of 5% phosphoric acid and the product was extracted withdichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered, andthen added to isopropyl alcohol. The precipitated product was removed byfiltration and dried under reduced pressure.

Yield 95.0 g. ¹H NMR (d₆-DMSO): δ1.72 ppm (q, CH₂ —CH₂—COO—), 2.24 ppm(t, —CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100% (meaningthat, within the accuracy of the NMR method, each OH— group located onthe ends of each arm of the 4-ARM-PEG starting material was converted tothe corresponding butanoic acid).

Example 2 Synthesis of 4-ARM-PEG_(20K)-Butanoate-Linked DocetaxelConjugate (“4-ARM-PEG_(20K)-BA-DOC”)

To a solution of 4-ARM-PEG_(20K)-butanoic acid (5.0 g, 0.0010equivalents, Example 1), docetaxel (1.0 g, 0.0012 moles), andp-toluenosulfonic acid 4-dimethylaminopyridine salt (0.15 g, 0.00051moles) in 60 ml of anhydrous dichloromethane,N,N′-diisopropylcarbodiimide (0.63 g, 0.005 mole) was added and themixture was stirred overnight at room temperature under an argonatmosphere. The solvent was distilled off under reduced pressure. Theresidue was dissolved in dichloromethane (7.5 ml) and added to a 1:1mixture of isopropyl alcohol and diethyl ether (100 ml). Theprecipitated product was filtered off and dried under reduced pressure.The precipitation was repeated giving 3.8 g of white solid product.

¹H NMR (400 MHz, CDCl₃): δ 1.06 (s, 12H), 1.16 (s, 12H), 1.27 (s, 36H),1.68 (s, 12H), 1.84 (m, 8H), 2.20-2.60 (m, 14H), 3.30-3.80 (m, ˜1900H),3.90 (d, 4H), 4.12 (d, 4H), 4.20 (m, 4H), 4.26 (d, 4H), 4.97 (d, 4H),5.15 (s, 4H), 5.28 (s, 4H), 5.38 (m, 8H), 5.62 (d, 4H), 6.30 (t, 4H),7.24 (m, 8H), 7.30 (m, 8H), 7.44 (m, 8H), 7.54 (m, 4H), 8.04 (d, 8H).

NMR analysis of the product in CDCl₃ as a solvent showed that to eachmolecule of 4ARM-PEG_(20K)-butanoic acid (4-ARM-PEG_(20K)-BA) wasconnected 4 molecules of docetaxel. Substitution: ˜98-100%.

Example 3 Synthesis of 4-ARM-PEG_(20K)-Glycine(Pentaerythritolyl-4-ARM-(PEG-1-Methylene-2 Oxo-Vinylamino Aceticacid)-20K) (“4-ARM-PEG_(20K)-CM-GLY”)

The following example is provided as a basis for comparing the extent ofdrug (i.e., docetaxel) substitution on the 4-ARM-PEG-OH startingmaterial described in Example 1, 4-ARM-PEG_(20K)-butanoic acid, versus4-ARM-PEG_(20K)-CM-glycine.

A. Formation of 4-ARM-PEG_(20K)-Acetic Acid (4-ARM-PEG_(20K)-CM)

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (100.0 g, 0.020equivalents) (NOF Corporation) in toluene (750 ml) was azeotropicallydried by distilling off 150 ml of toluene. 1.0M solution of potassiumtert-butoxide in tert-butanol (60 ml, 0.060 moles) and tert-butylbromoacetate (12.9 g, 0.066 moles) were added and the mixture wasstirred overnight at 45° C. under argon atmosphere. The solvent wasdistilled off under reduced pressure and the residue was dissolved indistilled water (1,000 ml). The pH of the solution was adjusted to 12 byaddition of 1M sodium hydroxide and the solution was stirred overnightkeeping the pH at 12.0 by periodic addition of 1M sodium hydroxide. ThepH was adjusted to 2 by addition of 1M phosphoric acid. The resultingproduct, 4-ARM-PEG_(20K)-acetic acid (also referred to as“4-ARM-PEG_(20K)-CM”) was extracted with dichloromethane. The extractwas dried over anhydrous sodium sulfate, filtered, and concentrated. Theconcentrated extract was then added to ethyl ether.

The precipitated product was collected by filtration and dried underreduced pressure. Yield 95.5 g. ¹H NMR (d₆-DMSO): δ 3.51 ppm (s, PEGbackbone), 4.01 ppm (s, —CH₂—COO—). Substitution 100%.Substitution=˜100% (meaning that, within the accuracy of the NMR method,each OH— group located on the ends of each arm of the 4-ARM-PEG startingmaterial was converted to the corresponding acetic acid).

B. Formation of 4-ARM-PEG_(20K)-Acetic Acid, N-Hydroxysuccinimide ester(NHS)

4-ARM-PEG_(20K)-acetic acid (90.0 g, 0.018 equivalents) was dissolved indichloromethane (270 ml) and N-hydroxysuccinimide (2.20 g, 0.019 mol)was added. Next dicyclohexylcarbodiimide (4.13 g, 0.020 moles) wasadded, and the solution was stirred at room temperature overnight. Thereaction mixture was filtered, concentrated, and precipitated byaddition to isopropyl alcohol.

Yield of final product: 82 g. ¹H NMR (d₆-DMSO): δ 2.81 ppm (s,succinimide), 3.51 ppm (s, PEG backbone), 4.60 ppm (s, —CH₂—COO—).Substitution 100%.

C. Formation of 4-ARM-PEG_(20K)-CM-Glycine (“4-ARM-PEG_(20K)-CM-GLY”)

4-ARM-PEG_(20K)-CM-Glycine

4-ARM-PEG_(20K)-acetic acid, N-hydroxysuccinimide ester (80.0 g, 0.016equivalents) was dissolved in dichloromethane (240 ml) and glycine,tert-butyl ester hydrochloride (3.22 g, 0.019 moles) was added. Nexttriethylamine (6.90 g) was added, and the solution was stirred at roomtemperature overnight. The reaction mixture was concentrated bydistilling off under reduced pressure 160 ml of dichloromethane,followed by addition of 80 ml of trifluoroacetic acid. The mixture wasstirred for three hours at room temperature, followed by distillationunder reduced pressure to remove dichloromethane and trifluoroaceticacid. The crude product was dissolved in 120 ml dichloromethane andprecipitated by addition of isopropyl alcohol.

The precipitate, (4-ARM-PEG_(20K)-CM-GLY) was collected and dried toprovide 74 g of a white solid product. ¹H NMR (CDCl₃): δ 3.64 ppm (s,PEG backbone), 4.05 ppm (s, —CH₂—COO—), 4.09 ppm (d, —CH₂—, glycine).Substitution ˜100%.

Example 4 Synthesis of 4-ARM-PEG_(20K)-CM-Glycine-linked DocetaxelConjugate (“4-ARM-PE-PEG_(20K)-CM-GLY-DOC”)

To a solution of 4-ARM-PEG_(20K)-acetate linked glycine(4-ARM-PEG_(20K)-CM-GLY, 5.0 g, 0.0010 equivalents), docetaxel (1.0 g,0.0012 moles), and p-toluenosulfonic acid 4-dimethylaminopyridine salt(1.5 g, 0.0051 moles) in 60 ml of anhydrous dichloromethane,N,N′-diisopropylcarbodiimide (0.63 g, 0.005 mole) was added and themixture was stirred overnight at room temperature under argonatmosphere. The solvent was distilled off under reduced pressure. Theresidue was dissolved in dichloromethane (7.5 ml) and added to a 1:1mixture of isopropyl alcohol and diethyl ether (100 ml). Theprecipitated product was filtered off and dried under reduced pressure.The precipitation was repeated giving 3.7 g of white solid product.

NMR analysis of the product (4-ARM-PEG_(20K)-CM-GLY-DOC) in CDCl₃ as asolvent showed that substitution was ˜75%. This means that each moleculeof 4ARM-PEG_(20K)-CM-GLY was connected to ˜3 molecules of docetaxelrather than the desired 4.

Example 5 Synthesis of Additional 4-ARM-PEG_(10K)-CM-Glycine-linkedDocetaxel Conjugates: (“4-ARM-PE-PEG_(10K)-CM-GLY-DOC”)

4-ARM-PEG_(10K)-CM-glycine-linked docetaxel conjugates were prepared ina similar fashion as described above, with the exception of theirmolecular weights, the average molecular mass of starting pentaerythitolbased 4-ARM-PEG-OH was 10 kDa.

¹H NMR analysis of the 4ARM-PEG_((10k))-glycine-linked docetaxelindicated ˜84% substitution.

Example 6 Synthesis of 4-ARM-PEG_(20K)-TEG-α-Methylpropionic Acid(“4-ARM-PEG_(20K)-TEG-α-MPA”)

I. Synthesis of tetra(ethylene glycol)-(α-amine, sulfuric acidsalt)-ω-(α-methylpropionic acid, methyl ester) A. Tetra(ethyleneglycol)mono-α-methylpropionitrile

A mixture of tetra(ethylene glycol), “TEG” (194.2 g, 1.0 mole),tetrabutylammonium bromide (9.6 g), and toluene (350 ml) wasazeotropically dried by distilling off toluene under reduced pressure.Solid potassium hydroxide (powder, 2.2 g) was added and the mixture wasstirred for 30 minutes at room temperature. Next methacrylonitrile (50ml) was added dropwise during two hours and the reaction mixture wasstirred for 92 hours at room temperature under argon atmosphere. Thecrude product was dissolved in 1500 ml deionized water and the resultingsolution was filtered through active carbon (50 g) and desalting columncomposed from Amberlite IR 120 plus (500 ml) and Amberlite IR 67. Sodiumchloride (300 g) was added and the product was extracted withdichloromethane (500, 300, and 200 ml). The extract was dried (MgSO₄)and the solvent was distilled off under reduced pressure.

Yield: 54.0 g. ¹H NMR (D2O): δ1.21 ppm (d, CH₃ —C—), 3.00 ppm (m,—CH—CN), 3.62 ppm (bm, —OCH₂CH₂O—). Purity: ˜100%.

B. Tetra(ethylene glycol)mono-α-methylpropionamide

Tetra(ethylene glycol)mono-α-methylpropionitrile (54 g) was dissolved inconcentrated hydrochloric acid (180 ml) and the resulting solution wasstirred for 20 hours at room temperature. Deionized water (250 ml) wasadded and the pH was adjusted to 7.6 with 10% solution of sodiumhydroxide at 7-8° C. Water was distilled under reduced pressure and theproduct was extracted from solid residue with anhydrous ethyl alcohol(200 ml). Ethyl alcohol was distilled from the extract and the crudeproduct was dissolved in dichloromethane (150 ml). The solution wasdried with anhydrous MgSO₄ and the solvent was distilled off underreduced pressure.

Yield: 53.3 g. ¹H NMR (D₂O): δ□0.89 ppm (d, CH₃ —C—), 2.55 ppm (m,—CH—(C═O)NH₂), 3.47 ppm (bm, —OCH₂CH₂O—). Purity: ˜100%.

C. Tetra(ethylene glycol)mono-α-methylpropionic acid

Tetra(ethylene glycol)mono-α-methylpropionamide (45 g) was dissolved inpotassium hydroxide solution (concentration 6.67%, 450 ml) and theresulting solution was stirred 70 h at room temperature. Next the pH ofthe reaction mixture was adjusted to 7.5 with 10% phosphoric acid attemperature 7-8° C. Water was distilled under reduced pressure and theproduct was extracted from solid residue with anhydrous ethyl alcohol(300 ml). Then ethyl alcohol was distilled from the extract. The crudeproduct was dissolved in deionized water (50 ml) and impurities wereextracted with dichloromethane (4×100 ml). NaCl (10 g) was added and thepH of the solution was adjusted to 3.0 with 10% hydrochloric acid. Theproduct was extracted with dichloromethane (100, 100, and 80 ml). Theextract was dried with anhydrous MgSO₄ and the solvent was distilled offunder reduced pressure.

Yield: 25.3 g. ¹H NMR (D₂O): δ□ 1.06 ppm (d, CH₃ —C—), 2.73 ppm (m,—CH—COO—), 3.60 ppm (bm, —OCH₂CH₂O—). Purity: ˜100%.

D. Tetra(ethylene glycol)mono-α-methylpropionic acid, methyl ester

Tetra(ethylene glycol)mono-α-methylpropionic acid (25 g) was dissolvedin anhydrous methyl alcohol (350 ml), concentrated sulfuric acid (6.5ml) was added and the solution was stirred for three hours at roomtemperature. The pH of the reaction mixture was adjusted to 6.0 with 8%NaHCO₃ aqueous solution and methyl alcohol was distilled off underreduced pressure. The product was extracted with dichloromethane (100,80, and 50 ml). The extract was dried with anhydrous MgSO₄ and thesolvent was distilled off under reduced pressure.

Yield: 24.0 g. ¹H NMR (d6-DMSO): δ□1.06 ppm (d, CH₃ —C—), 2.69 ppm (m,—CH—COO), 3.47 ppm (bm, —OCH₂CH₂O—), 360 ppm (s, CH₃O—). Purity: ˜100%.

E. Tetra(ethylene glycol)-α-mesylate-ω-(-α-methylpropionic acid, methylester)

A mixture of tetra(ethylene glycol)mono-α-methylpropionic acid, methylester (24 g, 0.0815 moles) and toluene (240 ml) was azeotropically driedby distilling off toluene under reduced pressure. The driedtetra(ethylene glycol)mono-α-methylpropionic acid, methyl ester wasdissolved in anhydrous toluene (200 ml). To the solution were added 40ml of anhydrous dichloromethane and 12.5 ml of triethylamine (0.0897moles). Then 10.0 g of methanesulfonyl chloride (0.0873 moles) dissolvedin dichloromethane (50 ml) was added dropwise at 0˜5° C. The solutionwas stirred at room temperature under argon atmosphere overnight. Nextsodium carbonate (10 g) was added, the mixture was stirred for one hour.Then the solution was filtered and solvents were distilled off underreduced pressure.

Yield: 28.3 g. ¹H NMR (d₆-DMSO): 1.06 ppm (d, CH₃—C—) 2.70 ppm (m,—CH—COO—), 3.18 ppm (s, CH₃—, methanesulfonate), 3.49 ppm (bm,—OCH₂CH₂O—), 3.60 ppm (s, CH₃O—), 3.67 ppm (m, —CH₂—CH₂-methanesulfonate), 4.31 ppm (m, —CH₂-methanesulfonate). Purity:˜100%.

F. Tetra(ethylene glycol)-(α-amine, hydrochloride)-ω-(-α-methylpropionicacid)

To a mixture of tetra(ethylene glycol)-α-mesylate-ω-(-α-methylpropionicacid, methyl ester) (23.8 g) and deionized water (50 ml), 70 ml of 5-%NaOH solution was added gradually during for six hours keeping the pH at12.0-12.3. Next concentrated ammonium hydroxide (1500 ml) was added andthe mixture was stirred for 64 hours at room temperature. Then themixture was concentrated to dryness under reduced pressure. The residuewas dissolved in deionized water (150 ml) and the pH of the solution wasadjusted to 3.0 with 1 M HCl. Water was distilled off under reducedpressure and the crude product was dissolved in dichloromethane (150ml). The solution was filtered, dried with anhydrous MgSO₄, and thesolvent was distilled off under reduced pressure.

Yield: 19.4 g. ¹H NMR (D₂O): 1.06 ppm (d, CH₃—C—) 2.72 ppm (m,—CH—COO—), 3.13 ppm (t, —CH₂ —NH₂*HCl), 3.64 ppm (bm, —OCH₂CH₂O—).Purity: ˜100%.

G. Tetra(ethylene glycol)-(α-amine, sulfuric acidsalt)-ω-(-α-methylpropionic acid, methyl ester)

Tetra(ethylene glycol)-(α-amine, hydrochloride)-ω-(-α-methylpropionicacid) (19.4 g) was dissolved in anhydrous methyl alcohol (280 ml),concentrated sulfuric acid (5.2 ml) was added and the solution wasstirred for three hours at room temperature. The pH of the reactionmixture was adjusted to 6.0 with 8% NaHCO₃ aqueous solution and thesolvents were distilled off under reduced pressure. The crude productwas dissolved in dichloromethane (250 ml). The solution was dried withanhydrous MgSO₄ and the solvent was distilled off under reducedpressure.

Yield: 15.7 g. ¹H NMR (D₂O): 1.04 ppm (d, CH₃—C—) 2.77 ppm (m,—CH—COO—), 3.11 ppm (t, —CH₂ —NH₂*H₂SO₄), 3.62 ppm (bm, —OCH₂CH₂O—).Purity: ˜100%.

II. Synthesis of 4-ARM-PEG_(20K)-Benzotriazolyl Carbonate(“4-ARM-PEG_(20K)-BTC”)

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (10.0 g, 0.0020equivalents) (NOF Corporation) in acetonitrile (100 ml) wasazeotropically dried by distilling off the solvent. Next the product wasredissolved in 50 ml of anhydrous acetonitrile and pyridine (0.49 ml)and di(1-benzotriazolyl)carbonate (1.31 g of 59% dispersion intrichloroethane) was added and the mixture was stirred at roomtemperature under nitrogen atmosphere overnight. The solvent wasdistilled off under reduced pressure and the product was precipitatedwith isopropyl alcohol. The precipitated product was dissolved indichloromethane (10 ml) and reprecipitated with isopropyl alcohol,filtered off and dried under reduced pressure.

Yield: 9.8 g. ¹H NMR (d₆-DMSO): 3.51 ppm (s, polymer backbone), 4.62 ppm(m, PEG-OCH₂—CH₂ —OCO₂—, 2H), 7.41-8.21 ppm (complex mult, benzotriazoleprotons, 4H). Substitution=˜100% (meaning that, within the accuracy ofthe NMR method, each OH— group located on the ends of each arm of the4-ARM-PEG starting material was converted to the correspondingbenzotriazolyl carbonate group).

III. Synthesis of 4-ARM-PEG_(20K)-TEG-α-Methylpropionic Acid(“4-ARM-PEG_(20K)-TEG-α-MPA”)

To a solution of tetra(ethylene glycol)-(α-amine, sulfuric acidsalt)-ω-(-α-methylpropionic acid, methyl ester) (0.75 g) in anhydrousdichloromethane (80 ml), triethylamine (0.56 ml) was added and thensolid 4-ARM-PEG_(20K)-benzotriazolyl carbonate (“4-ARM-PEG_(20K)-BTC”)(8.0 g) was added portionwise and the resulting solution was stirredovernight at room temperature under argon atmosphere. Next the solutionwas concentrated under reduced pressure and the product was precipitatedwith isopropyl alcohol and dried under vacuum. Next it was dissolved indistilled water (200 ml). The pH of the solution was adjusted to 12.1with 1M sodium hydroxide, and the solution was stirred for five hourskeeping the pH at 12.1 by periodic addition of 1M sodium hydroxide.Thereafter, NaCl (10 g) was added and the pH was adjusted to 3 byaddition of 10% phosphoric acid and the product was extracted withdichloromethane. The extract was dried over anhydrous sodium sulfate,filtered, and concentrated. The concentrated extract was then added toisopropyl alcohol.

The precipitated product was collected by filtration and dried underreduced pressure. Yield: 7.4 g. ¹H NMR (d₆-DMSO): δ 1.00 ppm (d,CH₃—C—), 2.55 ppm (m, —CH—COO—), 3.08 ppm (q, —CH₂N—NH—), 3.51 ppm (s,PEG backbone), 4.00 ppm (s, —CH₂—COO—), 7.16 ppm (t, —(C═O)—NH—).Substitution ˜100% (meaning that each OH— group located on the ends ofeach arm of the 4-ARM-PEG starting material was converted to thecorresponding □-methyl propionic acid).

Example 7 Synthesis of 4-ARM-PEG_(20K)-Hexanoic Acid(“4-ARM-PEG_(20K)-HA”)

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (50.0 g, 0.010equivalents) (NOF Corporation) in anhydrous toluene (500 ml) wasazeotropically dried by distilling off toluene. The dried product wasdissolved in anhydrous toluene (500 ml) and 1.0 M solution of potassiumtert-butoxide in tert-butanol (30 ml, 0.030 moles) and1-(5-bromopentyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (7.8 g,0.028 moles) were added and the mixture was stirred overnight at 72° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (400 ml). ThepH of the solution was adjusted to 2 with 10% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12.5 by addition of 1M sodium hydroxide, and thesolution was stirred for two hours keeping the pH at 12.1-12.5 byperiodic addition of 1M sodium hydroxide. Thereafter, the pH wasadjusted to 3 by addition of 10% phosphoric acid and the product wasextracted with dichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered,concentrated under reduced pressure and then added to ethyl ether. Theprecipitated product was removed by filtration and dried under reducedpressure.

Yield: 48.5 g. ¹H NMR (d₆-DMSO): δ 1.28 ppm (m, —CH₂—CH₂ —CH₂ —CH₂—CH₂—COO—), 1.48 ppm (m, —CH₂—CH₂—CH₂ —CH₂—CH₂—COO—), 2.18 ppm (t,—CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100% (meaning thateach OH— group located on the ends of each arm of the 4-ARM-PEG startingmaterial was converted to the corresponding hexanoic acid).

Example 8 Synthesis of 4-ARM-PEG_(20K)-Octanoic Acid(“4-ARM-PEG_(20K)-OA”)

I. Synthesis of 8-Bromooctanoate Ester of 3-Methyl-3-Oxetanemethanol

To a solution of 8-bromooctanoic acid (22.5 g, 0.101 moles),3-methyl-3-oxetanemethanol (11.0 g, 0.108 moles), and1-hydroxybenzotriazole (1.6 g) in anhydrous dichloromethane (500 ml)cooled to 0-5° C., a solution of N,N′-dicycloxehyldicarbodiimide (22.3g, 0.108 moles) in anhydrous dichloromethane (110 ml) was added dropwiseduring 45 minutes. Next, 4-(dimethylamino)pyridine (1.8 g) was added andthe mixture was stirred overnight at room temperature under argonatmosphere. The solution was filtered and washed two times with 5-%phosphoric acid (2×250 ml). Next it was dried with anhydrous MgSO4 andthe solvent was distilled off. Yield of crude product 28.0 g.

II. Synthesis of1-(7-bromoheptyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane

8-Bromooctanoate ester of 3-methyl-3-oxetanemethanol (14.0 g) wasdissolved in anhydrous dichloromethane (70 ml) and the solution wascooled to 0-5° C. Boron trifluoride diethyl etherate (1.49 ml) was addedand the mixture was stirred for four hours at 0-5° C. Triethylamine (7.2ml) was added and the mixture was stirred for 15 minutes at 0-5° C. Thesolvent was distilled off under reduced pressure. The residue wasdissolved in ethyl ether (200 ml) and the solution was filtered. Nextthe solvent was distilled off under reduced pressure. Yield 13.5 g.

III. Synthesis of 4-ARM-PEG_(20K)-Octanoic Acid

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (20.0 g, 0.004equivalents) (NOF Corporation) in anhydrous toluene (200 ml) wasazeotropically dried by distilling off toluene. The dried product wasdissolved in anhydrous toluene (400 ml) and 1.0 M solution of potassiumtert-butoxide in tert-butanol (12 ml, 0.012 moles) and1-(7-bromoheptyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (3.7 g,0.012 moles) were added and the mixture was stirred overnight at 72° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (300 ml). ThepH of the solution was adjusted to 2 with 10% phosphoric acid and thesolution was stirred for 45 minutes at room temperature. Next, the pHwas readjusted to 12.5 by addition of 1M sodium hydroxide, and thesolution was stirred for three hours keeping the pH at 12.1-12.5 byperiodic addition of 1M sodium hydroxide. Thereafter, the pH wasadjusted to 3 by addition of 10% phosphoric acid and the product wasextracted with dichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered,concentrated under reduced pressure and then precipitated with isopropylalcohol. The precipitated product was removed by filtration and driedunder reduced pressure, then it was dissolved in dichloromethane (15 ml)and reprecipitated with isopropyl alcohol. Yield: 15.5 g.

Yield: 48.5 g. ¹H NMR (d₆-DMSO): δ 1.25 ppm (m, —CH₂—CH₂ -CH₂—CH₂—CH₂-CH₂ —CH₂—COO—), 1.47 ppm (m, —CH₂—CH₂—CH₂—CH₂—CH₂ —CH₂—CH₂—COO—), 2.15 ppm (t, —CH₂—COO—), 3.51 ppm (s, PEG backbone).Substitution=˜100% (meaning that each OH— group located on the ends ofeach arm of the 4-ARM-PEG starting material was converted to thecorresponding octanoic acid).

Example 9 Synthesis of 4-ARM-PEG_(20K)-Decanoic Acid(“4-ARM-PEG_(20K)-DA”)

I. Synthesis of 10-Bromodecanoate Ester of 3-Methyl-3-Oxetanemethanol

To a solution of 10-bromooctanoic acid (25.0 g, 0.100 moles),3-methyl-3-oxetanemethanol (10.9 g, 0.107 moles), and1-hydroxybenzotriazole (1.6 g) in anhydrous dichloromethane (500 ml)cooled to 0-5° C., a solution of N,N′-dicycloxehyldicarbodiimide (22.1g, 0.107 moles) in anhydrous dichloromethane (110 ml) was added dropwiseduring 45 minutes. Next 4-(dimethylamino)pyridine (1.8 g) was added andthe mixture was stirred overnight at room temperature under argonatmosphere. The solution was filtered and washed two times with 5-%phosphoric acid (2×250 ml). Next it was dried with anhydrous MgSO4 andthe solvent was distilled off Yield 29.0 g.

II. Synthesis of1-(9-bromononyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane

10-Bromodecanoate ester of 3-methyl-3-oxetanemethanol (29.0 g) wasdissolved in anhydrous dichloromethane (300 ml) and the solution wascooled to 0-5° C. Boron trifluoride diethyl etherate (2.83 ml) was addedand the mixture was stirred for four hours at 0-5° C. Triethylamine(13.7 ml) was added and the mixture was stirred for 15 minutes at 0-5°C. The solvent was distilled off under reduced pressure. The residue wasdissolved in ethyl ether (400 ml) and the solution was filtered. Nextthe solvent was distilled off under reduced pressure. Yield 28.0 g.

III. Synthesis of 4-ARM-PEG_(20K)-Decanoic Acid

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (20.0 g, 0.004equivalents) (NOF Corporation) in anhydrous toluene (200 ml) wasazeotropically dried by distilling off toluene. The dried product wasdissolved in anhydrous toluene (400 ml) and 1.0 M solution of potassiumtert-butoxide in tert-butanol (12 ml, 0.012 moles) and1-(9-bromononyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (4.0 g, 0.012moles) were added and the mixture was stirred overnight at 72° C. underargon atmosphere. The solvent was distilled off under reduced pressureand the residue was dissolved in distilled water (300 ml). The pH of thesolution was adjusted to 2 with 10% phosphoric acid and the solution wasstirred for 45 minutes at room temperature. Next, the pH was readjustedto 12.5 by addition of 1M sodium hydroxide, and the solution was stirredfor three hours keeping the pH at 12.1-12.5 by periodic addition of 1Msodium hydroxide. Thereafter, the pH was adjusted to 3 by addition of10% phosphoric acid and the product was extracted with dichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered,concentrated under reduced pressure and then precipitated with isopropylalcohol. The precipitated product was removed by filtration and driedunder reduced pressure, then it was dissolved in dichloromethane (15 ml)and reprecipitated with isopropyl alcohol. Yield: 15.5 g.

¹H NMR (d₆-DMSO): δ 1.24 ppm (m, —CH₂—CH₂ —CH₂ —CH₂ —CH₂ —CH₂ —CH₂ —CH₂—CH₂—COO—), 1.46 ppm (m, —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂ —CH₂—CH₂—COO—),2.14 ppm (t, —CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100%(meaning that each OH— group located on the ends of each arm of the4-ARM-PEG starting material was converted to the corresponding decanoicacid).

Example 10 Synthesis of 4-ARM-PEG_(20K)-β-Methylpentanoic Acid(“4-ARM-PEG_(20K)-β-MPA”)

I. Synthesis of β-Methyl-δ-valerolactone

To a suspension of sodium borohydride (8.0 g) in anhydroustetrahydrofuran (160 ml), cooled to 0-5° C., a solution of3-methylglutaric anhydride (25.6 g) in anhydrous tetrahydrofuran (80 ml)was added and the mixture was stirred overnight at room temperature.Next, the mixture was cooled to 0-5° C. and hydrochloric acid (20%solution, 80 ml) was added during 40 minutes. The mixture was stirredfor 15 minutes at 0-5° C. and at four hours h at room temperature, nextit was concentrated under reduced pressure, filtered, and the crudeproduct was extracted with chloroform. The extract was dried withanhydrous MgSO₄ and the solvent was distilled under reduced pressure.

The crude product was purified by vacuum distillation collecting thefraction boiling at 78° C. at 2.6 mm Hg. Yield 13.1 g. NMR ˜100% pureproduct.

II. Synthesis of 5-Bromo-3-Methylpentanoic Acid

A solution of β-methyl-δ-valerolactone (3.0 g) in anhydrousdichloromethane (30 ml), cooled to 0-5° C., was saturated with hydrogenbromide gas and the mixture was stirred for three days at roomtemperature. The mixture was diluted with dichloromethane (100 ml) andthe resulting solution was washed with 10-% NaCl (3×30 ml). Next thesolution was dried with anhydrous MgSO₄ and the solvent was distilledunder reduced pressure, Yield: 4.6 g. NMR ˜100% pure product.

III. Synthesis of 5-Bromo-3-Methylpentanoate Ester of3-Methyl-3-Oxetanemethanol

To a solution of 5-bromo-3-methylpentanoic acid (4.6 g, 0.024 moles),3-methyl-3-oxetanemethanol (2.65 g, 0.026 moles), and1-hydroxybenzotriazole (0.40 g) in anhydrous dichloromethane (100 ml)cooled to 0-5° C., a solution of N,N′-dicycloxehyldicarbodiimide (5.35g, 0.026 moles) in anhydrous dichloromethane (20 ml) was added dropwiseduring 5 min. Next 4-(dimethylamino)pyridine (0.045 g) was added and themixture was stirred overnight at room temperature under argonatmosphere. The solution was filtered and washed two times with 5-%phosphoric acid (2×50 ml). Next it was dried with anhydrous MgSO₄ andthe solvent was distilled off. Yield 5.4 g. NMR purity ˜95%.

IV. Synthesis of 1-(4-bromo-2-methylbutyl)-4-methyl-2,6,7trioxabicyclo[2,2,2]octane

5-Bromo-3-methylpentanoate ester of 3-methyl-3-oxetanemethanol (5.4 g)was dissolved in anhydrous dichloromethane (400 ml) and the solution wascooled to 0-5° C. Boron trifluoride diethyl etherate (0.7 ml) was addedand the mixture was stirred for four hours at 0-5° C. Triethylamine (3.4ml) was added and the mixture was stirred for 15 minutes at 0-5° C. Thesolvent was distilled off under reduced pressure. The residue wasdissolved in ethyl ether (50 ml) and the solution was filtered. Next thesolvent was distilled off under reduced pressure. Yield: 4.6 g.

V. Synthesis of 4-ARM-PEG_(20K)-α-Methylpentanoic Acid

A solution of pentaerythitol-based 4-ARM-PEG_(20K)-OH (7.5 g, 0.0015equivalents) (NOF Corporation) in anhydrous toluene (37.5 ml) wasazeotropically dried by distilling off toluene. The dried product wasdissolved in anhydrous toluene (37.5 ml) and 1.0 M solution of potassiumtert-butoxide in tert-butanol (9 ml, 0.0090 moles) and1-(4-bromo-2-butyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (2.3 g,0.0083 moles) were added and the mixture was stirred overnight at 70° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (100 ml). ThepH of the solution was adjusted to 2 with 10% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12.5 by addition of 1M sodium hydroxide, and thesolution was stirred for three hours keeping the pH at 12.1-12.5 byperiodic addition of 1M sodium hydroxide. Thereafter, the pH wasadjusted to 3 by addition of 10% phosphoric acid and the product wasextracted with dichloromethane. The extract was dried over anhydrousmagnesium sulfate, filtered, concentrated under reduced pressure andthen precipitated with isopropyl alcohol. The precipitated product wasremoved by filtration and dried under reduced pressure. Yield: 3.2 g.

¹H NMR (D₂O): δ 0.88 ppm (d, —CH₃), 1.44 ppm and 1.56 ppm (m, .—CH₂ —CH₂-√{square root over (CH(CH₃))}—CH₂—COO—), 1.93 ppm (m, CH—CH₃), 2.11 ppmand 2.31 ppm (m, —CH₂—COO—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEGbackbone). Substitution=˜75.5%.

Example 11 Characterization of Conjugates

The extent of drug substitution for various multi-arm conjugates ofdocetaxel is summarized below, as determined by H NMR.

Drug Substitution: Calculation of Drug Substitution by ¹H NMR

Samples of different 4-ARM-PEG-Docetaxel conjugates in deuteratedchloroform (CDCl₃) were analyzed by ¹H NMR, and the integral of thepolymer backbone was compared with that of aromatic protons of docetaxelat 8.10 ppm. Based upon the averaged proton peak integration of allspectra obtained, the extent of drug substitution was determined.

TABLE 1 Conjugate NMR % (percent substitution) 4-ARM-PEG_(20K)-GLY-DOC(74% or on average 3 molecules drug per multi-arm polymer)4-ARM-PEG_(10K)-GLY-DOC (81% or on average 3.2 molecules drug permulti-arm polymer) 4-ARM-PEG_(20K)-BA-DOC (95%* or on average 3.8molecules drug per multi-arm polymer) *Drug substitution values averageabout 98% substitution for the BA-linked conjugate,4-ARM-PEG_(20K)-BA-DOC (95-100%).

Example 12 Synthesis of Additional Multi-Armed Polymer Alkanoate-LinkedDocetaxel Conjugates

The following 4-ARM-PEG_(20K)-alkanoic acids were prepared as describedin Example 1: 4-ARM-PEG_(20K)-α-methylpropionic acid(4-ARM-PEG_(20K)-MPA), 4-ARM-PEG_(20K)-hexanoic acid(4-ARM-PEG_(20K)-HA), 4-ARM-PEG_(20K)-octanoic acid(4-ARM-PEG_(20K)-OA), and 4-ARM-PEG_(20K)-decanoic acid(4-ARM-PEG_(20K)-DA). The following multi-armed butanoic acids were alsoprepared: 4-ARM-PEG_(10K)-butanoic acid (4-ARM-PEG_(10K)-BA),4-ARM-PEG_(30K)-butanoic acid (4-ARM-PEG_(30K)-BA), and4-ARM-PEG_(40K)-butanoic acid (4-ARM-PEG_(40K)-BA). Detaileddescriptions of the synthesis follow as Examples 12A-C respectively.

Docetaxel (1.03 g, 1.20 mmol), the corresponding 4-ARM-PEG_(20K)-Acid[α-methylpropionic acid (4-ARM-PEG_(20K)-MPA), 4-ARM-PEG_(20K)-hexanoicacid (4-ARM-PEG_(20K)-HA), 4-ARM-PEG_(20K)-octanoic acid(4-ARM-PEG_(20K)-OA), 4-ARM-PEG_(20K)-decanoic acid(4-ARM-PEG_(20K)-DA), 4-ARM-PEG_(10K)-butanoic acid(4-ARM-PEG_(10K)-BA), 4-ARM-PEG_(30K)-butanoic acid(4-ARM-PEG_(10K)-BA), or 4-ARM-PEG_(40K)-butanoic acid(4-ARM-PEG_(40K)-BA), 0.25 mmol], and DPTS (0.15 g, 0.50 mmol) weredissolved in 50 mL DCM. The resulting solution was cooled in an ice-salt(NaCl) bath (−15 to −5° C.) for 20 minutes. before DIC (0.38 g, 3.00mmol, in 5 mL of DCM) was added with stirring. The reaction mixture wasstirred for an additional 12 hours at −15 to −5° C. The solution wasfiltered to remove any solids and then concentrated under reducedpressure to half the volume. The resulting solution was slowly added to400 mL of ether/IPA (1:1) with stirring. The white solid was collectedand precipitated again using the above method. The product was dried invacuo (yield: approximately 90% for all syntheses).

4-ARM-PEG_(20K)-MPA-Docetaxel: ¹H NMR (300 MHz, CDCl₃): δ 1.02-1.20 (m,24H), 1.21 (s, 12H), 1.32 (s, 36H), 1.65 (d, 4H), 1.74 (s, 12H), 1.85(m, 4H), 1.93 (s, 12H), 2.10-2.38 (m, 8H), 2.41 (s, 12H), 2.60 (m, 4H),2.80 (m, 4H), 3.30-3.92 (m, ˜2180H), 4.10-4.30 (m, 23H), 4.95 (d, 4H),5.21 (s, 4H), 5.47 (m, 4H), 5.52 (br., 10H), 6.21 (t, 4H), 7.26 (m, 8H),7.40 (m, 8H), 7.50 (m, 8H), 7.60 (m, 4H), 8.12 (d, 8H).

4-ARM-PEG_(20K)-HA-Docetaxel: ¹H NMR (300 MHz, CDCl₃): δ 1.12 (s, 12H),1.22 (s, 12H), 1.32 (s, 36H), 1.34-1.70 (m, 15H), 1.74 (s, 12H),1.80-1.95 (m, 16H), 2.10-2.38 (m, 16H), 2.43 (s, 12H), 2.60 (m, 4H),3.30-3.90 (m, ˜1950H), 3.91 (d, 4H), 4.12-4.26 (m, 8H), 4.30 (d, 4H),4.97 (d, 4H), 5.28 (s, 4H), 5.25-5.55 (m, 10H), 5.68 (d, 4H), 6.21 (t,4H), 7.26 (m, 8H), 7.40 (m, 8H), 7.50 (m, 8H), 7.60 (m, 4H), 8.11 (d,8H).

4-ARM-PEG_(20K)-OA-Docetaxel ¹H NMR (300 MHz, CDCl₃): δ 1.11 (s, 12H),1.13-1.24 (m, 40H), 1.33 (s, 36H), 1.40-1.60 (m, 18H), 1.74 (m, 12H),1.83-2.00 (m, 16H),), 2.00-2.20 (m, 4H), 2.220-2.40 (m, 13H), 2.43 (s,12H), 2.60 (br., 4H), 3.32-3.89 (m, ˜2180H), 3.90 (d, 4H), 4.12-4.20 (m,12H), 4.26 (d, 4H), 4.96 (d, 4H), 5.21 (s, 4H), 5.38 (m, 8H), 5.50 (br.4H), 5.68 (d, 4H), 6.23 (t, 4H), 7.24 (m, 8H), 7.38 (m, 8H), 7.50 (m,8H),), 7.61 (m, 4H), 8.12 (d, 8H).

4-ARM-PEG_(20K)-DA-Docetaxel ¹H NMR (300 MHz, CDCl₃): δ 1.11 (s, 12H),1.22 (s, 12H), 1.23-1.30 (m, 44H), 1.33 (s, 36H), 1.40-1.60 (m, 18H),1.74 (m, 12H), 1.84-2.00 (m, 16H), 2.00-2.20 (m, 4H), 2.20-2.40 (m,13H), 2.43 (s, 12H), 2.60 (br., 4H), 3.30-3.88 (m, ˜2100H), 3.90 (d,4H), 4.12-4.20 (m, 12H), 4.26 (d, 4H), 4.96 (d, 4H), 5.21 (s, 4H), 5.38(m, 8H),), 5.50 (br. 4H), 5.68 (d, 4H), 6.23 (t, 4H), 7.24 (m, 8H), 7.38(m, 8H), 7.50 (m, 8H), 7.61 (m, 4H), 8.12 (d, 8H).

4-ARM-PEG_(10K)-BA-Docetaxel ¹H NMR (500 MHz, CDCl₃): δ 1.10 (s, 12H),1.21 (s, 12H), 1.33 (s, 36H), 1.74 (s, 12H), 1.85 (m, 12H), 1.94 (m,12H), 2.15 (m, 4H), 2.30 (m, 4H), 2.41 (m, 15H), 2.55 (m, 8H), 3.30-3.80(m, ˜960H), 3.90 (d, 4H), 4.18 (m, 8H), 4.25 (m, 4H), 4.30 (d, 4H), 4.97(d, 4H), 5.19 (s, 4H), 5.30 (s, 4H), 5.45 (m, 8H), 5.64 (d, 4H), 6.30(t, 4H), 7.24 (m, 8H), 7.35 (m, 8H), 7.49 (m, 8H), 7.60 (m, 4H), 8.01(d, 8H).

4-ARM-PEG_(30K)-BA-Docetaxel ¹H NMR (500 MHz, CDCl₃): δ 1.11 (s, 12H),1.21 (s, 12H), 1.33 (s, 36H), 1.74 (s, 12H), 1.85 (m, 12H), 1.94 (m,12H), 2.15 (m, 4H), 2.30 (m, 4H), 2.41 (m, 15H), 2.55 (m, 8H), 3.30-3.80(m, ˜2840H), 3.90 (d, 4H), 4.18 (m, 8H), 4.25 (m, 4H), 4.30 (d, 4H),4.97 (d, 4H), 5.19 (s, 4H), 5.30 (s, 4H), 5.45 (m, 8H), 5.64 (d, 4H),6.30 (t, 4H), 7.24 (m, 8H), 7.35 (m, 8H), 7.49 (m, 8H), 7.60 (m, 4H),8.01 (d, 8H).

4-ARM-PEG_(40K)-BA-Docetaxel ¹H NMR (500 MHz, CDCl₃): δ 1.11 (s, 12H),1.21 (s, 12H), 1.33 (s, 36H), 1.74 (s, 12H), 1.85 (m, 12H), 1.94 (m,12H), 2.15 (m, 4H), 2.30 (m, 4H), 2.41 (m, 15H), 2.55 (m, 8H), 3.30-3.80(m, ˜4050H), 3.90 (d, 4H), 4.18 (m, 8H), 4.25 (m, 4H), 4.30 (d, 4H),4.97 (d, 4H), 5.19 (s, 4H), 5.30 (s, 4H), 5.45 (m, 8H), 5.64 (d, 4H),6.30 (t, 4H), 7.24 (m, 8H), 7.35 (m, 8H), 7.49 (m, 8H), 7.60 (m, 4H),8.09 (d, 8H).

Example 12A Synthesis of 4-ARM-PEG_(10K)-Butanoic Acid(“4-ARM-PEG_(10K)-BA”)

A solution of pentaerythritol-based 4-ARM-PEG_(10K)-OH (50.0 g, 0.020equivalents) (NOF Corporation) in toluene (450 ml) was azeotropicallydried by distilling off 100 ml of toluene. A 1.0M solution of potassiumtert-butoxide in tert-butanol (50 ml, 0.050 moles) and1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (14.0 g,0.056 moles) was added and the mixture was stirred overnight at 70° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (600 ml). ThepH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 by addition of 1M sodium hydroxide, and thesolution was stirred for two hours keeping the pH at 12 by periodicaddition of 1M sodium hydroxide. Thereafter, the pH was adjusted to 3 byaddition of 5% phosphoric acid and the product was extracted withdichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered, andthen added to isopropyl alcohol. The precipitated product was removed byfiltration and dried under reduced pressure.

Yield: 45.0 g. ¹H NMR (d₆-DMSO): δ 1.72 ppm (q, CH₂ —CH₂—COO—), 2.24 Ppm(t, —CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100% (meaningthat each OH— group located on the end of each arm of the 4-ARM-PEGstarting material was converted to the corresponding butanoic acid).

Example 12B Synthesis of 4-ARM-PEG_(30k)-Butanoic Acid(“4-ARM-PEG_(30K)-BA”)

A solution of pentaerythritol-based 4-ARM-PEG_(30k)-OH (50.0 g, 0.0067equivalents) (ChemOrganics, Houston Tex.) in toluene (450 ml) wasazeotropically dried by distilling off 100 ml of toluene. A 1.0Msolution of potassium tert-butoxide in tert-butanol (20 ml, 0.020 moles)and 1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (5.9 g,0.023 moles) was added and the mixture was stirred overnight at 70° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (600 ml). ThepH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 by addition of 1M sodium hydroxide, and thesolution was stirred for two hours keeping the pH at 12 by periodicaddition of 1M sodium hydroxide. Thereafter, the pH was adjusted to 3 byaddition of 5% phosphoric acid and the product was extracted withdichloromethane,

The extract was dried over anhydrous magnesium sulfate, filtered, andthen added to isopropyl alcohol. The precipitated product was removed byfiltration and dried under reduced pressure.

Yield: 46 g. ¹H NMR (d₆-DMSO): δ 1.72 ppm (q, CH₂ —CH₂—COO—), 2.24 ppm(t, —CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100% (meaningthat each OH— group located on the ends of each arm of the 4-ARM-PEGstarting material was converted to the corresponding butanoic acid).

Example 12C Synthesis of 4-ARM-PEG_(40k)-Butanoic Acid(“4-ARM-PEG_(40K)-BA”)

A solution of pentaerythritol-based 4-ARM-PEG_(40K)-OH (50.0 g, 0.005equivalents) (NOF Corporation) in toluene (450 ml) was azeotropicallydried by distilling off 100 ml of toluene. A 1.0M solution of potassiumtert-butoxide in tert-butanol (15 ml, 0.015 moles) and1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (4.4 g,0.018 moles) was added and the mixture was stirred overnight at 70° C.under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (600 ml). ThepH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 by addition of 1M sodium hydroxide, and thesolution was stirred for two hours keeping the pH at 12 by periodicaddition of 1M sodium hydroxide. Thereafter, the pH was adjusted to 3 byaddition of 5% phosphoric acid and the product was extracted withdichloromethane.

The extract was dried over anhydrous magnesium sulfate, filtered, andthen added to isopropyl alcohol. The precipitated product was removed byfiltration and dried under reduced pressure.

Yield: 48.0 g. ¹H NMR (d₆-DMSO): δ 1.72 ppm (q, CH₂ —CH₂—COO—), 2.24 ppm(t, —CH₂—COO—), 3.51 ppm (s, PEG backbone). Substitution=˜100% (meaningthat each OH— group located on the ends of each arm of the 4-ARM-PEGstarting material was converted to the corresponding butanoic acid).

Example 13 Anti-Tumor Activity of 4-ARM-PEG_(70K)-BA-docetaxel in MouseXenograft Models of Human Lung and Colon Cancer

The purpose of the following studies was to evaluate the anti-tumoractivity of 4-ARM-PEG_(20K)-BA-DOC in non-small cell lung (H460) andcolorectal (LS174T, LoVo) mouse tumor xenograft models exhibitinglimited docetaxel sensitivity.

H460, LS174T and LoVo tumors were established in female athymic nudemice. Groups of 10 mice received a total of three single doses of4-ARM-PEG_(20K)-BA-DOC or docetaxel administered every 7 days (q7dx3) atseveral doses up to a maximum tolerated dose (MTD). Control groupsreceived no treatment after tumor implant. Animals were weighed dailyfor the first five days and twice weekly thereafter. Tumor volumes weremeasured twice weekly after the first drug injection.

Tumor endpoint volume was 1500 mm³ for LS174T, 1000 mm³ for LoVo, and2000 mm³ for H460.

Anti-tumor activity was measured as changes in tumor volume, time toreach endpoint, and complete or partial tumor regression.

Treatment outcome was evaluated by Tumor Growth Delay (TGD), which isdefined as the increase in median time to endpoint in a treatment groupcompared to the control group. Here it is expressed as % TGD, as apercentage to the median time to endpoint of the control group.

A tumor whose volume was reduced to 50% or less for three consecutivemeasurements compared to its volume on Day 1 was considered to be inpartial regression. Complete regression was defined as volume equal toor greater than 13.5 mm³ for three consecutive measurements.

Acceptable toxicity was defined as a group mean body weight (BW) loss ofless than 20% during the study and not more than one treatment-relateddeath among ten treated animals. Any dosing regimen that resulted ingreater toxicity was considered above the maximum tolerated dose (MTD).

Results were as follows.

Non-Small Cell Lung Cancer Model (H460):

4-ARM-PEG_(20K)-BA-DOC treatment resulted in a statistically significant(p<0.05), dose-related increase in tumor growth delay (TGD) in H460tumor bearing mice at MTD (33 mg/kg 4-ARM-PEG_(20K)-BA-DOC, 25 mg/kgdocetaxel) with % TGD values of 122% and 48% in 4-ARM-PEG_(20K)-BA-DOCand docetaxel treated groups, respectively. See FIGS. 3A (all dosagegroups) and 3B (control and MTD groups only) as well as Table 2A below.Decreases in body weight were comparable between 4-ARM-PEG_(20K)-BA-DOCand docetaxel at MTD (14.2% and 15.5%, respectively).

To assess whether the improved antitumor activity was due to greaterplasma and tumor docetaxel exposure following 4-ARM-PEG_(20K)-BA-DOCadministration, plasma and tumor homogenates were analyzed after asingle IV administration of 4-ARM-PEG_(20K)-BA-DOC or docetaxel in micebearing H460 tumors. In looking at each of 4-ARM-PEG_(20K)-BA-DOC anddocetaxel dosed at their respective maximum tolerated doses (MTDs) of 33and 25 mg/kg, the following was observed. After 4-ARM-PEG_(20K)-BA-DOCadministration, plasma docetaxel concentrations remained above 5 ng/mLfor as long as 168 hr, whereas docetaxel concentrations fell below 1ng/mL by 72 hr after docetaxel administration. The maximum plasmadocetaxel concentration with 4-ARM-PEG_(20K)-BA-DOC was 13-fold lowerthan that with docetaxel. While both treatments achieved a similarplasma docetaxel area under the concentration-time curve (AUC),4-ARM-PEG_(20K)-BA-DOC treatment resulted in tumor docetaxelconcentrations that were 0.4 to 4-fold higher than those for docetaxel,leading to a 2-fold greater tumor docetaxel AUC. This greater andsustained tumor exposure to docetaxel after 4-ARM-PEG_(20K)-BA-DOCadministration was associated with significantly longer tumor growthdelay over docetaxel (122% vs 48%). 4-ARM-PEG_(20K)-BA-DOC retainedsignificant antitumor activity at 19 mg/kg (58% of its MTD), whereasdocetaxel doses less than 25 mg/kg did not.

TABLE 2A Response Summary Following Treatment with4-ARM-PEG_(20K)-BA-DOC and Docetaxel in H460 Xenografts BW Loss No.Surviving/ Treatment Dose Dose TGD¹ (%)/Day of No. Treated Group (mg/kg)(mg/m²) (%) BW Nadir Mice No Treatment 0 0 —  0/0² 10/10 Docetaxel 10 30−2 0/0 10/10 14 42 4 4.1/19  10/10 18.7 56.1 23 9.7/22  10/10 25 75  48*15.5/26   10/10 4-ARM- 14 42 −1 0/0 10/10 PEG_(20K)- 18.7 56.1  56*6.2/22  10/10 BA-DOC 25 75   72** 10.4/29   10/10 33.3 99.9   122**14.2/22   10/10 *p < 0.05 as compared to no treatment group. **p < 0.01as compared to no treatment group. ¹Tumor growth delay is based on astudy endpoint of either 2000 mm³ or 57-days. ²0/0 indicates no decreasein body weight was observed. ³Statistical significance was notevaluated, since treatment exceeded MTD.

TABLE 2B 4-ARM-PEG_(20K)-BA- Tumor AUC DOC to Docetaxel growth delay(hr*ng/mL) Ratio Treatment Dose (mg/kg)^(&) (%) Plasma Tumor PlasmaTumor Docetaxel 25 MTD 48 6146 138,715 — — 4-ARM-PEG_(20K)- 33 MTD 1226013 313,558 1.0 2.3 BA-DOC 19 58% 56  3488^(#)  181,864^(#) 0.6 1.3 MTD^(&)Administered q7dx3 ^(#)Extrapolated from the 33 mg/kg dose.

Colon (LS174T) Cancer Model:

TGD with 4-ARM-PEG_(20K)-BA-DOC treatment in LS174T tumor bearing micewas dose-related and resulted in three partial regressions (PR) at MTD;no partial regressions were observed with docetaxel. In LS174T tumorbearing mice, 4-ARM-PEG_(20K)-BA-DOC demonstrated a significantly(p<0.0001) greater TGD than docetaxel; 266% vs. 166% at MTD (40 mg/kg4-ARM-PEG_(20K)-BA-DOC, 30 mg/kg docetaxel). At 30 mg/kg,4-ARM-PEG_(20K)-BA-DOC demonstrated a 250% TGD and one partialregression while 30 mg/kg docetaxel resulted in 166% TGD and noregressions. Decreases in body weight were comparable between4-ARM-PEG_(20K)-BA-DOC and docetaxel at MTD (18.8% and 16.6%,respectively.

See FIGS. 2A and 2B and Table 3 below.

TABLE 3 Response Summary Following Treatment with 4-ARM-PEG_(20K)-BA-DOCand Docetaxel in LS174T Colon Cancer Xenografts BW Loss No. (%)/DaySurviving/ Treatment Dose Dose TGD ¹ of BW No. Treated Group (mg/kg)(mg/m²) (%) Nadir PR Mice No 0 0 —    0/0 ² 0 10/10 Treatment 12.7 38.1 69*  8.8/23 0 10/10 Docetaxel 16.9 50.7  96* 11.2/23 0 10/10 22.5 67.5135*  6.7/23 0 10/10 30 90 166* 16.6/23 0 10/10 4-ARM- 16.9 50.7 126*12.4/23 0  9/10 PEG_(20K)-BA- 22.5 67.5 169* 11.1/23 0 10/10 DOC 30 90250* 17.9/23 1  9/10 40 120 266* 18.8/23 3 10/10 *p < 0.001 as comparedto no treatment group. ¹ TGD based on a study endpoint of either 1500mm³ or 79-days. ² 0/0 indicates no decrease in body weight was observed.

Colon (LoVo) Cancer Model:

4-ARM-PEG_(20K)-BA-DOC treatment in LoVo tumor bearing mice resulted insimilar dose-related improvements in TGD. Body weight changes for4-ARM-PEG_(20K)-BA-DOC treatment were generally less than or similar tothose for docetaxel at the same equivalent dose. 40 mg/kg4-ARM-PEG_(20K)-BA-DOC was in excess of the MTD due to a body weightdecrease of 22.6% and resulted in a 148% TGD with two PRs. Although notsignificantly different, when administered at a docetaxel equivalentdose of 30 mg/kg, the MTD for both agents, 4-ARM-PEG_(20K)-BA-DOCresulted in twice the tumor growth delay compared to docetaxel (128% vs64%). At the lower docetaxel equivalent doses (22.5 and 16.9 mg/kg),activity of 4-ARM-PEG_(20K)-BA-DOC and docetaxel was comparable. SeeFIGS. 4A and 4B.

TABLE 4 Response Summary Following Treatment with 4-ARM-PEG_(20K)-BA-DOC and Docetaxel in LoVo Xenografts BW Loss No. (%)/DaySurviving/ Treatment Dose Dose TGD ² of BW No. Treated Group (mg/kg)(mg/m²) (%) Nadir PR Mice No 0 0 —    0/0 ³ 0 10/10 Treatment 12.7 38.17  5.0/24 0 10/10 Docetaxel 16.9 50.7  70** 12.8/21 0 10/10 22.5 67.532* 14.6/24 0 10/10 30 90  64** 13.3/21 0 10/10 4-ARM- 16.9 50.7  64** 7.0/21 0 10/10 PEG_(20K)-BA- 22.5 67.5 50* 11.0/24 0 10/10 DOC 30 90128** 16.2/24 0 10/10 40 120 148 ¹  22.6/24 2 10/10 *p < 0.01 ***p <0.001 as compared to no treatment group. ¹ Statistical significance wasnot evaluated, since treatment exceeded MTD. ² TGD based on a studyendpoint of 1000 mm³ or 77-days. ³ 0/0 indicates no decrease in bodyweight was observed.

The foregoing illustrates that 4-ARM-PEG_(20K)-BA-DOC possessessignificantly greater anti-tumor activity than docetaxel in H460 and LS174T mouse xenograft models. Partial regressions were observed in two ofthe three cell lines for 4-ARM-PEG_(20K)-BA-DOC while no regressionswere observed for docetaxel. At MTD, the % TGDs for4-ARM-PEG_(20K)-BA-DOC were 2.5- and 1.6-fold greater than docetaxel inH460 and LS174 xenograft models, respectively. Further,4-ARM-PEG_(20K)-BA-DOC was well tolerated, with weight loss at MTDcomparable to docetaxel and no significant clinical observations. Insummary, the foregoing illustrates that the exemplary conjugate,4-ARM-PEG_(20K)-BA-DOC, is effective at significantly improving thetime-concentration profile and anti-tumor activity of docetaxel.

Example 14 Pharmacokinetics of Docetaxel and4-ARM-PEG_(20K)-BA-docetaxel in Sprague-Dawley Rats

The objective of this study was to assess the pharmacokinetics andexcretion of docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel afterintravenous infusion to rats.

Pharmacokinetics of 4-ARM-PEG_(20K)-BA-docetaxel and docetaxel wereevaluated in male rats (15 rats/dose level) dosed with 5, 10, and 15mg/kg (30, 60, 90 mg/m²) 4-ARM-PEG_(20K)-BA-docetaxel or 5 mg/kg (30mg/m²) docetaxel. 4-ARM-PEG_(20K)-BA-docetaxel and docetaxel wereadministered via a femoral vein catheter as 30-min intravenous infusionsat 4 mL/kg. Blood samples (n=15 timepoints with n=5 samples/doselevel/timepoint) were obtained by jugular venipuncture between pre-doseand 144 hours post-dose. Pharmacokinetic parameters are summarized inTable 5.

TABLE 5 Docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel PharmacokineticParameters in Male Rats after Single IV Doses of 5 mg/kg Docetaxel, or5, 10, 15 mg/kg 4-ARM-PEG_(20K)-BA-docetaxel Dose T_(1/2) Tmax CmaxAUClast AUCinf CL Vss Treatment (mg/kg) Analyte (hr) (hr) (ng/mL)(hr*ng/mL) (hr*ng/mL) (mL/min/kg) (L/kg) Docetaxel 5 Docetaxel   ND ¹0.5 682 1417 ND ND² ND Conjugate* 5 Docetaxel ND 0.5 122 1711 ND NA NAConjugate 73 0.5 40079 226203 250374 0.33 0.8  Conjugate* 10 DocetaxelND 2.0 176 3003 ND NA NA Conjugate 59 0.5 70795 419132 437912 0.38 0.55Conjugate* 15 Docetaxel  ND. 1.0 273 3743 ND NA NA Conjugate 63 0.5138296 847993 895159 0.28 0.47 Average Conjugate (±SD) 65 ± 7 0.33 ±0.05 0.61 ± 0.17 ¹ ND = not determined; terminal phase not well defined²NA = not applicable for the docetaxel metabolite *Conjugate =4-ARM-PEG_(20K)-BA-docetaxel

Plasma 4-ARM-PEG_(20K)-BA-docetaxel concentrations declined in amultiphasic fashion with an average terminal half-life of 65 hr.4-ARM-PEG_(20K)-BA-docetaxel clearance was low at 0.33 mL/min/kg. TheVss was similar to the total body water, indicating distribution outsidethe vascular space. T_(1/2), CL, and V_(ss) were independent of dose.C_(max) and AUC values increased in a dose-related and generallydose-proportional manner.

Plasma docetaxel concentrations declined in a multiphasic fashion andremained sustained after administration of 4-ARM-PEG_(20K)-BA-docetaxel.Docetaxel C_(max) values were reached between 0.5-2 hr after the4-ARM-PEG_(20K)-BA-docetaxel dose, indicating that a portion ofdocetaxel is released quickly. At equivalent doses (5 mg/kg or 30mg/mm²) 4-ARM-PEG_(20K)-BA-docetaxel, 4-ARM-PEG_(20K)-BA-docetaxeltreatment resulted in about a 6-fold lower C_(max) but similar AUC.Docetaxel C_(max) and AUC after 4-ARM-PEG_(20K)-BA-docetaxel increasedin a dose-related and generally dose-proportional manner.

Standard non-compartmental analysis did not allow accurate estimation ofthe long docetaxel terminal half-life after administration of4-ARM-PEG_(20K)-BA-docetaxel, due in part to the sustained presence ofdocetaxel and the variability associated with concentrations in theslowest disposition phase occurring near the lower limit ofquantification (1 ng/mL). To further evaluate the sustained plasmadocetaxel terminal half-life, a population pharmacokinetic approach wasemployed. The population pharmacokinetic analysis estimated a terminalhalf life for docetaxel of 168 hr after administration of4-ARM-PEG_(20K)-BA-docetaxel and 40 hr after administration ofdocetaxel.

Based upon the foregoing, 4-ARM-PEG_(20K)-BA-docetaxel when administeredto rats has a low clearance and low volume of distribution, resulting ina long 4-ARM-PEG_(20K)-BA-docetaxel terminal half life of 65 hr. Plasmadocetaxel C_(max) is approximately 6-fold lower after administration ofequivalent 4-ARM-PEG_(20K)-BA-docetaxel and docetaxel doses, while AUCis similar. Plasma docetaxel half-life is estimated to be about 4-foldlonger following 4-ARM-PEG_(20K)-BA-docetaxel administration than thatobserved following docetaxel administration (168 vs. 40 hr) resulting insustained systemic docetaxel exposure.

Example 15 Pharmacokinetics of Docetaxel and4-ARM-PEG_(20K)-BA-docetaxel in Beagle Dogs

The objective of the study was to assess the pharmacokinetics andexcretion of docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel afterintravenous infusion to dogs.

Pharmacokinetics were evaluated in male dogs (4 dogs/dose level) dosedwith 0, 0.75, 2, or 4 mg/kg (0, 15, 40, and 80 mg/m²/dose) of4-ARM-PEG_(20K)-BA-docetaxel or 0.75 mg/kg (15 mg/m²) docetaxel.4-ARM-PEG_(20K)-BA-docetaxel and docetaxel were administered via acephalic or saphenous vein disposable catheter as 60-min intravenousinfusions at 4 mL/kg/hr. Seventeen blood samples were obtained byjugular venipuncture or Abbacoth IV catheter between 0 and 168 hrpost-dose. Plasma concentrations of 4-ARM-PEG_(20K)-BA-docetaxel anddocetaxel were determined.

Pharmacokinetic parameters are summarized in Table 6.

TABLE 6 Docetaxel and 4-ARM-PEG20K-BA-Docetaxel PharmacokineticParameters in Male Dogs after Single IV Doses of 0.75 mg/kg Docetaxel,and 0.75, 2, and 4 mg/kg 4-ARM-PEG_(20K)-BA-docetaxel Dose T_(1/2) TmaxCmax AUClast AUCinf CL Vss Treatment (mg/kg) Analyte (hr) (hr) (ng/mL)(hr*ng/mL) (hr*ng/mL) (mL/min/kg) (L/kg) Docetaxel 0.75 Docetaxel   ND ¹0.88 223 479 ND ND ² ND Conjugate 0.75 Docetaxel ND 0.88 2.1 64 ND NA NAConjugate 28 1.1 7084 93084 104247 0.12 0.23 Conjugate 2 Docetaxel ND1.1 6.3 153 ND NA NA Conjugate 35 1.4 17731 232378 245446 0.14 0.26Conjugate 4 Docetaxel ND 12 19 326 ND NA NA Conjugate 32 6.8 33570597336 611470 0.12 0.31 Average Conjugate (±SD) 32 ± 4 0.13 ± 0.01 0.26± 0.04 ¹ ND = not determined; terminal phase not well defined ² NA = notapplicable for the docetaxel metabolite *Conjugate =4-ARM-PEG_(20K)-BA-docetaxel

Plasma 4-ARM-PEG_(20K)-BA-docetaxel concentrations declined in amultiphasic fashion with an average terminal half-life of 31 hr.4-ARM-PEG_(20K)-BA-docetaxel clearance was low at 0.13 mL/min/kg. TheVss was less than total body water, indicating limited distributionoutside the vascular space. T_(1/2), CL, and V_(ss) are independent ofdose. C_(max) and AUC values following 4-ARM-PEG_(20K)-BA-docetaxeladministration increased in a dose-related and generallydose-proportional manner.

Plasma docetaxel concentrations declined in a multiphasic fashion andremained sustained after administration of 4-ARM-PEG_(20K)-BA-docetaxel.Docetaxel C_(max) values are reached between 0.88-12 hr after the4-ARM-PEG_(20K)-BA-docetaxel dose, indicating that a portion ofdocetaxel is released quickly. At docetaxel and4-ARM-PEG_(20K)-BA-docetaxel equivalent dose levels of 0.75 mg/kg,4-ARM-PEG_(20K)-BA-docetaxel treatment resulted in a 110-fold lowerC_(max) and 7-fold lower AUC. Docetaxel Cmax and AUC increase in adose-related and generally dose-proportional manner.

Standard noncompartmental analysis did not allow accurate estimation ofthe long docetaxel terminal half-life after administration of4-ARM-PEG_(20K)-BA-docetaxel, due in part to the sustained presence ofdocetaxel and the variability associated with concentrations in theslowest disposition phase occurring near the lower limit ofquantification (1 ng/mL). To further evaluate the sustained plasmadocetaxel terminal half-life, a population pharmacokinetic approach wasemployed. The population pharmacokinetic analysis predicts a docetaxelterminal half life in dogs of 199 hours after administration of4-ARM-PEG_(20K)-BA-docetaxel, and 25 hr after administration ofdocetaxel.

Lower docetaxel AUC after 4-ARM-PEG_(20K)-BA-docetaxel is likely due toincomplete capture of AUC because of a relatively short sampling period(168 hours) in the presence of a 199-hour apparent half-life. Based onthis finding, it is likely that docetaxel release from4-ARM-PEG_(20K)-BA-docetaxel continues for a significant time afterdosing.

Based upon this study, 4-ARM-PEG_(20K)-BA-docetaxel has a low clearanceand low volume of distribution, resulting in a long4-ARM-PEG_(20K)-BA-docetaxel terminal half-life of 31 hr. Plasmadocetaxel C_(max) and AUC are about 110- and 7-fold lower, respectively,following administration of equivalent 4-ARM-PEG_(20K)-BA-docetaxel anddocetaxel doses. Lower docetaxel AUC after 4-ARM-PEG_(20K)-BA-docetaxelis likely due to incomplete capture of AUC because of a relatively shortsampling period (168 hours) in the presence of a 199-hour apparenthalf-life. Plasma docetaxel half-life following4-ARM-PEG_(20K)-BA-docetaxel administration is estimated to be about8-fold longer than that observed following docetaxel administration (199vs. 25 hours) confirming the sustained systemic docetaxel exposureobserved in rats.

Example 16 Nonlinear Mixed Effect Modeling of Terminal Disposition RateConstants

While determining the pharmacokinetics of docetaxel after administrationof docetaxel or 4-ARM-PEG_(20K)-BA-docetaxel, it was observed thatreliable values for the terminal disposition rate constant λ_(z) couldnot be obtained for the majority of animals secondary to fluctuation ofplasma docetaxel concentrations between adjacent sampling times and/orthe occurrence of incomplete concentration-time profiles due toconcentrations being near or at the lower limit of quantification (1ng/mL) In addition, in Example 14, rats were sampled in groups of 15using alternating, overlapping sparse sampling schemes across groups toencompass the total duration of sampling. All of these factorscontributed to the occurrence of sparse data within a given animal'sterminal concentration-time profile.

To further investigate and compare docetaxel terminal dispositionpatterns between docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel, populationpharmacokinetic methods were applied to data from rats (Example 14) anddogs (Example 15 and in one additional study). This allowed estimationof population λ_(z) values and corresponding half-life values for eachtreatment group in the presence of incomplete data within individualanimals. Specifically, data from all animals within a treatment groupand all treatment groups were fit with a nonlinear mixed effectpharmacokinetic model using the program Monolix. Plasma docetaxelconcentrations following docetaxel or 4-ARM-PEG_(20K)-BA-docetaxeladministration, and plasma 4-ARM-PEG_(20K)-BA-docetaxel concentrationsfollowing 4-ARM-PEG_(20K)-BA-docetaxel administration were fit with amodel specifying zero-order input of the dose over a defined infusionperiod and biexponential disposition (Monolix PK Model 26). Docetaxelconcentration-time data after 4-ARM-PEG_(20K)-BA-docetaxel were fit witha model having first order input, to reflect metabolism of4-ARM-PEG_(20K)-BA-docetaxel to the active docetaxel metabolite, andbiexponential disposition (Monolix PK Model 31). The parameter ofinterest for this investigation was the population terminal dispositionrate constant, X, to supplement the results described above.

A. Population Modeling Results for Docetaxel in Sprague-Dawley Rats

Plasma docetaxel concentration-time profiles in rats afteradministration of docetaxel or 4-ARM-PEG_(20K)-BA-docetaxel were wellfit with the respective models. It was possible to obtain a populationestimate for λ_(z) for all 4 treatment groups. The results show thatdocetaxel concentrations in the terminal disposition phase followingadministration of all doses of 4-ARM-PEG_(20K)-BA-docetaxel are moresustained than those following administration of docetaxel.

The population mean (SE) λ_(z) estimates for docetaxel followingadministration of docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel to rats are0.0174 (0.0018) hr⁻¹ and 0.00412 (0.001) hr⁻¹, respectively, withcorresponding t½_(λz) values of 40 and 168 h.

B. Population Modeling Results for Docetaxel in Beagle Dogs

Plasma docetaxel concentration-time profiles in dogs afteradministration of docetaxel or 4-ARM-PEG_(20K)-BA-docetaxel were wellfit with the respective models. It was possible to obtain a populationestimate for λ_(z) for all 4 treatment groups. The modeling supportsthat docetaxel concentrations in the terminal disposition phasefollowing administration of all doses of 4-ARM-PEG_(20K)-BA-docetaxelare more sustained than those following administration of docetaxel,consistent with the trend observed in rats.

The population mean (SE) λ_(z) estimates for docetaxel followingadministration of docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel to dogs are0.0276 (0.0022) hr⁻¹ and 0.00349 (0.0021) hr⁻¹, respectively, withcorresponding t½_(λz) values of 25 and 199 hr.

Dogs in the additional study received 7 mg/kg doses of4-ARM-PEG_(20K)-BA-docetaxel on Days 1 and 22 with serial blood samplingafter each dose for determination of plasma 4-ARM-PEG_(20K)-BA-docetaxeland docetaxel concentrations.

The population mean (SE) λ_(z) estimates for4-ARM-PEG_(20K)-BA-docetaxel and docetaxel following administration of4-ARM-PEG_(20K)-BA-docetaxel to dogs are 0.0141 (0.00041) h⁻¹ and0.00627 (0.00086) h⁻¹, respectively, with corresponding t½_(λz) valuesof 49 and 110 hr.

When compared within and across studies, population mean λ_(z) estimatesshow that plasma docetaxel concentrations are sustained followingadministration of 4-ARM-PEG_(20K)-BA-docetaxel relative toadministration of docetaxel, independent of species and dose.

TABLE 7 Summary of Docetaxel Population Mean λ_(z) Estimates andCorresponding t1/2λz Values Across Species and Studies. SpeciesTreatment Dose (mg/kg) Mean (SE) λ_(z) (hr⁻¹) t1/2_(λz) (hr) RatDocetaxel 5 0.0174 (0.0018) 40 Conjugate 5, 10, 15 0.00412 (0.001)   168Dog Docetaxel 0.75 0.0276 (0.0022) 25 Conjugate 0.72, 2, 4 0.00349(0.0021)  199 Dog Conjugate 7 0.00627 (0.00086) 110 *Conjugate =4-ARM-PEG_(20K)-BA-docetaxel

Values for docetaxel t½_(λz) after 4-ARM-PEG_(20K)-BA-docetaxel are 4-to 8-fold greater than those after docetaxel in rats and dogs, providingevidence of the desired characteristics of prolonged docetaxel half-lifeand extended exposure.

Example 17 Pharmacokinetic Study of 4-ARM-PEG_(20K)-BA-DOCETAXEL inFemale Athymic Nude Mice Implanted Subcutaneously with H460 NSCLC TumorFragments

The objective of this study was to compare docetaxel pharmacokineticsfollowing 4-ARM-PEG_(20K)-BA-docetaxel with that following docetaxeladministration, in plasma and tumor tissue of athymic female micebearing H460 NSCLC tumors and to determine the effects on complete bloodcounts. This example summarizes the pharmacokinetic results.

H460 tumor cells were grown in vitro and implanted subcutaneously intofemale HRLN nude mice. When tumor sizes reached between 172 and 288 mg,animals were dosed by intravenous bolus injection with 25 mg/kgdocetaxel or 33 mg/kg 4-ARM-PEG_(20K)-BA-docetaxel at a dosing volume of10 mL/kg. These dose levels correspond to the maximum tolerated dosesfor docetaxel and 4-ARM-PEG_(20K)-BA-docetaxel administered on a q7dx3schedule in this tumor model. Blood and tumor samples (n=7 timepointswith n=3/timepoint) were collected from 0 to 7 days after IV injection.Plasma and tumor samples were analyzed for docetaxel and4-ARM-PEG_(20K)-BA-docetaxel.

Both 4-ARM-PEG_(20K)-BA-docetaxel lots resulted in comparable plasma andtumor drug concentration time profiles. 4-ARM-PEG_(20K)-BA-docetaxeldistributes to and remains in the tumor tissue, resulting in a tumor toplasma ratio of about 7 by 168 hr post-dose.

Plasma docetaxel concentrations fell below the limit of quantitation (1ng/mL) by 72 hr after administration of docetaxel, while they remainedabove 5 ng/mL for up to 168 hr after administration of4-ARM-PEG_(20K)-BA-docetaxel. These results demonstrate sustainedsystemic exposure to docetaxel after 4-ARM-PEG_(20K)-BA-docetaxeladministration in a third animal species. Both lots of4-ARM-PEG_(20K)-BA-docetaxel resulted in comparable plasma and tumordocetaxel concentration-time profiles.

Plasma and tumor 4-ARM-PEG_(20K)-BA-docetaxel and docetaxel exposuredata are summarized in Table 8.

TABLE 8 Plasma and Tumor Exposures after Administration of 25 mg/kgDocetaxel and 33 mg/kg 4-ARM-PEG_(20K)-BA-docetaxel to H460-Bearing NudeMice Conjugate to Dose Tmax Cmax AUClast Docetaxel ratio Treatment(mg/kg) Analyte Compartment (hr) (ng/mL) (hr*ng/mL) Cmax AUC Docetaxel25 Docetaxel Plasma 0.5 2571 6146 Tumor 4 2336 138715 Conjugate 33Conjugate Plasma 0.5 128354 1097606 Lot 1 Tumor 4 7062 652823 DocetaxelPlasma 12 195 6013 0.08 1.0 Tumor 12 2931 313558 1.3 2.3 Conjugate 33Conjugate Plasma 0.5 118209 1852510 Lot 2 Tumor 4 7675 718068 DocetaxelPlasma 12 162 5570 0.06 0.9 Tumor 12 4623 380617 2.0 2.7 *Conjugate =4-ARM-PEG_(20K)-BA-docetaxel

Plasma 4-ARM-PEG_(20K)-BA-docetaxel Cmax was reached in 0.5 hr post-dose(first timepoint measured). Tumor Cmax was reached by 4 hr post-dose.4-ARM-PEG_(20K)-BA-docetaxel Cmax was 17-fold and AUC approximately2-fold lower in tumor compared to plasma.

Plasma docetaxel Cmax was reached in 0.5 hr post-dose (first timepointmeasured) independent of treatment. Tumor docetaxel Cmax was reached by4 hr post docetaxel dose and 12 hr post 4-ARM-PEG_(20K)-BA-docetaxeldose. Plasma docetaxel Cmax was reduced about 14-fold afteradministration of 4-ARM-PEG_(20K)-BA-docetaxel, but the sustained plasmadrug concentrations after 4-ARM-PEG_(20K)-BA-docetaxel resulted insimilar mean docetaxel AUC values after 4-ARM-PEG_(20K)-BA-docetaxel forboth treatments. The rate of decline in tumor docetaxel concentrationsappeared similar after administration of docetaxel and4-ARM-PEG_(20K)-BA-docetaxel, although tumor docetaxel concentrationsafter 4-ARM-PEG_(20K)-BA-docetaxel were approximately 5-fold higher inthe period from 24 hr post dose through the end of sampling,demonstrating greater and sustained tumor docetaxel exposure.

The greater and sustained tumor docetaxel exposure after4-ARM-PEG_(20K)-BA-docetaxel correlated with a significantly longer H460tumor growth delay for 4-ARM-PEG_(20K)-BA-docetaxel vs. docetaxel (122vs. 48%).

This study provides evidence that the superior antitumor activity of4-ARM-PEG_(20K)-BA-docetaxel is mediated by increased and sustainedtumor docetaxel exposure.

Example 18 Stability of 4-ARM-PEG_(20K)-BA-DOCETAXEL in Mouse, Rat, Dogand Human Plasma

The objective of the study was to assess the stability of4-ARM-PEG_(20K)-BA-docetaxel in mouse, rat, dog, and human plasma.

4-ARM-PEG_(20K)-BA-docetaxel was spiked (50 μg/mL; 60 μM) intoheparinized, pooled male mouse, rat, and dog plasma; mixed sex pooledhuman plasma; and phosphate buffered saline. Triplicate samples wereincubated at 37° C. for 0, 15, 30, 60, 120, and 240 min. Aliquots wereremoved at each timepoint and analyzed for disappearance of4-ARM-PEG_(20K)-BA-docetaxel and appearance of docetaxel using thevalidated dog plasma analytical method.

Table 9 summarizes the disappearance of 4-ARM-PEG_(20K)-BA-docetaxel andappearance of docetaxel at the end of the 240-minute incubation period.

TABLE 9 Plasma Stability of 4-ARM-PEG_(20K)-BA-docetaxel afterIncubation with Buffer, Mouse, Rat, Dog and Human Plasma Mouse Rat DogHuman Buffer Conjugate* remaining at 4 hr (%) 72 64 93 88 85 Docetaxelreleased at 4 hr (%) ¹ 20 20 3 4 0.6 Mass balance (%) 92 84 95 92 86 ¹Mean (n = 3), expressed as a percentage of starting conjugateconcentrations. ² Mean (n = 3), expressed as a percentage of totaldocetaxel content. *Conjugate = 4-ARM-PEG_(20K)-BA-docetaxel

Docetaxel release from 4-ARM-PEG_(20K)-BA-docetaxel was detected inplasma from all 4 species after 4 hr of incubation. Docetaxel percentreleased was 20% in mouse and rat plasma but notably less and similar indog (3%) and human plasma (4%). This finding is consistent with reportsthat rodents have more esterases with greater esterase activity thannon-rodent species (Williams F. M., Clin. Pharmacokinet. 1985: 10:392-403, Kaliste-Korhonen E, et al., Human & Experimental Toxicology1996: 15: 972-978, Li, B., et al., Biochem. Pharmacol 2005: 70:1673-1684), and that rodents typically metabolize ester containing drugsand prodrugs at greater rates and to a greater extent (Li, B., et al.,Biochem. Pharmacol 2005: 70: 1673-1684; Cook C. S., et al., Pharm. Res.1995: 12: 1158-1164; Quon C. Y., et al., Drug Metabolism and Disposition1988: 16: 425-428; Minagawa T, et al., Biochem. Pharmacol. 1995: 49:1361-1365; Ericsson H., et al., Eur. J. Pharm. Sci. 1999: 8: 29-37). Theresults of this study are consistent with reports that metabolism ofester containing drugs and prodrugs by rodents is not necessarilyrepresentative or predictable of that in humans.

Docetaxel is released from 4-ARM-PEG_(20K)-BA-docetaxel after incubationin mouse, rat, dog, and human plasma. The release of docetaxel isnotably faster in rodent plasma than in non-rodent plasma, suggestingthat rodents are less reliable than the dog as predictors of humanmetabolism of 4-ARM-PEG_(20K)-BA-docetaxel.

Example 19 Dose Escalation Study of Exemplary Multi-Armed PolymerAlkanoate Linked Docetaxel Conjugates

This study was undertaken to investigate the effect of polymer size andlinker identity on the toxicity of the conjugate. Docetaxel conjugatesas described in Example 12 were investigated to determine their maximumtolerated dose (MTD) when administered to Sprague-Dawley rats as asingle dose.

Healthy rats were randomized into eighteen groups of five male and 5females per group. Weight variation of the animals did not exceed ±10%of mean body weight. Prior to dosing, each of the test articles wasdissolved in 5% dextrose solution in sterile water for injection. Thearticles were each dosed as a single intravenous bolus injection inconscious rats. Dose volume was maintained at 2 mL/kg body weight.

A dose of 12.5 mg/kg (docetaxel equivalents) for all agents wasadministered first to groups of 5 male and 5 female rats. If themortality was 10% or less at 48 hours after dosing, a dose of 20 mg/kgof the test agent was administered to the next groups of 5 male and 5female rats. If mortality from the 12.5 mg/kg dose was greater than 10%,the second dose was subsequently modified. The third dose wasadministered based on the results from the first two doses and wasadjusted accordingly on the basis of the mortality results obtained.

Clinical observations were performed 15 minutes, 30 minutes, 1, 2, 3,and 4 hours after dosing and once post-noon observation on the day oftest article administration. The observations were continued twicedaily, once in the morning and in the afternoon on the remaining 14 dayobservation period.

Overall mortality was determined after 14 days observation at each doselevel. See the results in Table 10 below. As noted above, mortalitywithin first 48 hours of dosing was used to escalate to the next higherdose. Clinical signs before death included reduced locomotor activity,ataxic gait, polyurea, diarrhoea and emaciation.

TABLE 10 Dose DE 10K 10K 30K 30K 40K 40K 20K β 20K β 20K 20K (mg/kg) BABA BA BA BA BA MPA MPA OA OA — M F M F M F M F M F 12.5 0/5 0/5 2/5 1/54/5 4/5 0/5 0/5 0/5 0/5 20 0/5 0/5 4/5 5/5 5/5 5/5 0/5 0/5 0/5 0/5 250/5 0/5 5/5 5/5 5/5 5/5 0/5 0/5 0/5 0/5

In looking at the results, it can be seen that for the butanoate-linkedconjugates, preliminary toxicity data indicate that the relative orderof toxicity in rats, based upon polymer molecular weight, is:10K<20K<30K<40K. In comparing data over one polymer molecular weight(i.e., constant polymer size of 20K) versus linker structure, basedsolely on the rat data, it appears that the relative order of toxicityis MPA is about the same as OA<BA<HA.

The invention(s) set forth herein has been described with respect toparticular exemplified embodiments. However, the foregoing descriptionis not intended to limit the invention to the exemplified embodiments,and the skilled artisan should recognize that variations can be madewithin the spirit and scope of the invention as described in theforegoing specification.

1. A method of treating a mammalian subject for a condition responsiveto treatment with docetaxel, where the method comprises administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a multi-armed polymer conjugate having thestructure,

where n ranges from about 40 to about 500 or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable excipient. 2.The method of claim 1, wherein the weight average molecular weight ofthe conjugate is about 20,000 daltons.
 3. The method of claim 1, whereinthe condition responsive to treatment with docetaxel is selected fromthe group consisting of breast cancer, ovarian cancer, colon cancer,colorectal cancer, prostate cancer, gastric cancer, malignant melanoma,small cell lung cancer, non-small cell lung cancer, thyroid cancers,kidney cancer, cancer of the bile duct, brain cancer, cancer of the headand neck, lymphomas, leukemias, rhabdomyosarcoma, and neuroblastoma.